Aleksandra M. Mirończuk

Efficient conversion of crude glycerol from various industrial wastes into single cell oil by yeast Yarrowia lipolytica

In this study, crude glycerol from various industries was used to produce lipids via wild type Yarrowia lipolytica A101. We tested samples without any prior purification from five different waste products; each contained various concentrations of glycerol (42-87%) as the sole carbon source. The best results for lipid production were obtained for medium containing glycerol from fat saponification. This reached 1.69gL(-1) (25% of total cell dry weight) with a biomass yield of 0.17gg(-1) in the flasks experiment. The batch cultivation in a bioreactor resulted in enhanced lipid production-it achieved 4.72gL(-1) with a biomass yield 0.21gg(-1). Moreover, the properly selected batch of crude glycerol provides a defined fatty acid composition. In summary, this paper shows that crude glycerol from soap production could be efficiently converted to single cell oil without any prior purification.

A two-stage fermentation process of erythritol production by yeast Y. lipolytica from molasses and glycerol.

In this study, a two-stage fermentation process of erythritol production based on molasses and glycerol was investigated. During the first stage, the biomass of Yarrowia lipolytica was grown on medium containing sucrose as the sole carbon source. In the second stage, production of erythritol was initiated by glycerol addition. To use molasses as a substrate for erythritol synthesis, sucrose utilization was established by expressing the Saccharomyces cerevisiae SUC2 gene. In this study, cultivation of yeast Y. lipolytica could produce 52-114 g/L of erythritol. The productivity was 0.58-1.04 g/L/h, and yield was 0.26-0.57 g/g; the final biomasses yield ranged 17-41 g/L. This is the first report describing erythritol production via industrial raw molasses and glycerol by Y. lipolytica. This work uses genetically modified strains of Y. lipolytica as tool for the direct conversion of affordable raw industrial molasses and glycerol into the value-added erythritol product.

Polyol production from waste materials by genetically modified Yarrowia lipolytica

Sugar alcohols (polyols) are sweeteners with many industrial applications. In this study, a fermentation process of polyol production based on waste substrates - raw industrial molasses and crude glycerol - was tested. The yeast strain Yarrowia lipolytica Wratislavia K1 was genetically modified by overexpression of the Saccharomyces cerevisiae SUC2 gene and overexpression of the native GUT1 gene. This process allowed for sucrose utilization and rapid glycerol assimilation by the engineered strain. In this study, the obtained strain AIB pAD-UTGut1 produced 100.65±3.75g/l of polyols, with productivity of 1.09±0.9g/lh and yield of 0.67±0.2g/g. This is the first study describing efficient polyol production by the modified Y. lipolytica strain from industrial raw molasses and crude glycerol. By process optimization, we established conditions for abundant polyol synthesis from low-value substrates.

Recent advances in biological production of erythritol

Abstract Erythritol is a natural sweetener commonly used in the food and pharmaceutical industries. Produced by microorganisms as an osmoprotectant, it is an ideal sucrose substitute for diabetics or overweight persons due to its almost zero calorie content. Currently, erythritol is produced on an industrial scale through the fermentation of sugars by some yeasts, such as Moniliella sp. However, the popularity of erythritol as a sweetener is still small because of its high retail price. This creates an opportunity for further process improvement. Recent years have brought the rapid development of erythritol biosynthesis methods from the low-cost substrates, and a better understanding of the metabolic pathways leading to erythritol synthesis. The yeast Yarrowia lipolytica emerges as an organism effectively producing erythritol from pure or crude glycerol. Moreover, novel erythritol producing organisms and substrates may be taken into considerations due to metabolic engineering. This review focuses on the modification of erythritol production to use low-cost substrates and metabolic engineering of the microorganisms in order to improve yield and productivity.

EUF1 – a newly identified gene involved in erythritol utilization in Yarrowia lipolytica

The gene YALI0F01562g was identified as an important factor involved in erythritol catabolism of the unconventional yeast Yarrowia lipolytica. Its putative role was identified for the first time by comparative analysis of four Y. lipolytica strains: A-101.1.31, Wratislavia K1, MK1 and AMM. The presence of a mutation that seriously damaged the gene corresponded to inability of the strain Wratislavia K1 to utilize erythritol. RT-PCR analysis of the strain MK1 demonstrated a significant increase in YALI0F01562g expression during growth on erythritol. Further studies involving deletion and overexpression of the selected gene showed that it is indeed essential for efficient erythritol assimilation. The deletion strain Y. lipolytica AMM∆euf1 was almost unable to grow on erythritol as the sole carbon source. When the strain was applied in the process of erythritol production from glycerol, the amount of erythritol remained constant after reaching the maximal concentration. Analysis of the YALI0F01562g gene sequence revealed the presence of domains characteristic for transcription factors. Therefore we suggest naming the studied gene Erythritol Utilization Factor – EUF1.

A Role of a Newly Identified Isomerase From Yarrowia lipolytica in Erythritol Catabolism

Erythritol is a natural sweetener produced by microorganisms as an osmoprotectant. It belongs to the group of polyols and it can be utilized by the oleaginous yeast Yarrowia lipolytica. Despite the recent identification of the transcription factor of erythritol utilization (EUF1), the metabolic pathway of erythritol catabolism remains unknown. In this study we identified a new gene, YALI0F01628g, involved in erythritol assimilation. In silico analysis showed that YALI0F01628g is a putative isomerase and it is localized in the same region as EUF1. qRT-PCR analysis of Y. lipolytica showed a significant increase in YALI0F01628g expression during growth on erythritol and after overexpression of EUF1. Moreover, the deletion strain ΔF01628 showed significantly impaired erythritol assimilation, whereas synthesis of erythritol remained unchanged. The results showed that YALI0F1628g is involved in erythritol assimilation; thus we named the gene EYI1. Moreover, we suggest the metabolic pathway of erythritol assimilation in yeast Y. lipolytica.

An Effective Method of Continuous Production of Erythritol from Glycerol by Yarrowia lipolytica MK1

This study demonstrates the potential applicability of the UV mutant Yarrowia lipolytica MK1 for the valorisation of glycerol and erythritol production in a chemostat culture. The aim of this research is to investigate the optimal C:N ratio in the feeding medium in order to enhance erythritol production. The highest erythritol concentration, at 113.1 g/L with a volumetric erythritol production rate of 1.1 g/(L·h) and a yield of 0.57 g/g, was obtained in the feeding medium with a C:N ratio of 80:1. Moreover, no residual glycerol was observed in the culture broth during cultivation. The chemical composition of the biomass was analysed. The contents of lysine and threonine in the biomass protein amino acid profile were higher than those required by the FAO/WHO for fodder yeast.

Isolation and characterization of Arctic microorganisms decomposing bioplastics

The increasing amount of plastic waste causes significant environmental pollution. In this study, screening of Arctic microorganisms which are able to degrade bioplastics was performed. In total, 313 microorganisms were isolated from 52 soil samples from the Arctic region (Spitsbergen). Among the isolated microorganisms, 121 (38.66%) showed biodegradation activity. The ability of clear zone formation on emulsified poly(butylene succinate-co-adipate) (PBSA) was observed for 116 microorganisms (95.87%), on poly(butylene succinate) (PBS) for 73 microorganisms (60.33%), and on poly(ɛ-caprolactone) (PCL) for 102 microorganisms (84.3%). Moreover, the growth of microorganisms on poly(lactic acid) (PLA) agar plates was observed for 56 microorganisms (46.28%). Based on the 16S rRNA sequence, 10 bacterial strains which showed the highest ability for biodegradation were identified as species belonging to Pseudomonas sp. and Rhodococcus sp. The isolated fungal strains were tested for polycaprolactone films and commercial corn and potato starch bags degradation under laboratory conditions. Strains 16G (based on the analysis of a partial 18S rRNA sequence, identified as Clonostachys rosea) and 16H (identified as Trichoderma sp.) showed the highest capability for biodegradation. A particularly high capability for biodegradation was observed for the strain Clonostachys rosea, which showed 100% degradation of starch films and 52.91% degradation of PCL films in a 30-day shake flask experiment. The main advantage of the microorganisms isolated from Arctic environment is the ability to grow at low temperature and efficient biodegradation under this condition. The data suggest that C. rosea can be used in natural and laboratory conditions for degradations of bioplastics.

Aseptic production of citric and isocitric acid from crude glycerol by genetically modified Yarrowia lipolytica

The unconventional yeast Yarrowia lipolytica is known for its capacity to produce citric or isocitric acid from glycerol. In this study a reduction of production cost was achieved by using cheap crude glycerol and conducting the production at pH 3 to prevent bacterial contamination. In this study a Y. lipolytica strain overexpressing Gut1 and Gut2 was used. For the modified strain, crude glycerol proved to be an excellent substrate for production of citric/isocitric acids in aseptic conditions, as the final concentration of these compounds reached 75.9 ± 1.8 g L-1 after 7 days of batch production. Interestingly, the concentration of isocitric acid was 42.5 ± 2.4 g L-1, which is one of the highest concentrations of isocitric acid obtained from a waste substrate. In summary, these data show that organic acids can be efficiently produced by the yeast Y. lipolytica from crude glycerol without any prior purification in aseptic conditions.

Influence of ylHog1 MAPK kinase on Yarrowia lipolytica stress response and erythritol production

Erythritol production is a unique response to hyperosmotic stress that is observed in a small group of yeasts, including Yarrowia lipolytica. This study investigated whether this unusual mechanism is regulated by the HOG pathway, well described in Saccharomyces cerevisiae. The gene YALI0E25135g was identified as the Y. lipolytica homologue of HOG1 and was found to be phosphorylated in response to hyperosmotic shock. Deletion of the gene caused a significant decrease in resistance to hyperosmotic stress and negatively affected erythritol production. Interestingly, the deletion strain yl-hog1Δ displayed significant morphological defects, with the cells growing in a filamentous form. Moreover, yl-hog1Δ cells were also resistant to the cell wall damaging agents Congo red and calcofluor white. Collectively, these results indicate that yl-Hog1 is crucial for the cellular response to hyperosmotic stress, plays a role in the induction of erythritol production, and potentially prevents cross-talk with different MAPK signalling pathways in the cell.