Waste fat biodegradation and biomodification by Yarrowia lipolytica and a bacterial consortium composed of Bacillus spp. and Pseudomonas putida

Fats, oils and greases (FOGs) are a particular environmental threat. Biodegradation of FOGs is a challenge and in this study the biodegradation of waste cooking fats, namely butter and olive oil, was studied using a non-conventional yeast, Yarrowia lipolytica strain LFMB 20, and a bioaugmentation product consisting of Bacillus spp. and Pseudomonas putida CP1 strain. The microorganisms were grown aerobically in shake-flask experiments in an enriched medium supplemented with ca 0.85% w/v of waste fat. Analysis of the remaining substrate showed a removal of ca 90% of the fat by the yeast at the end of the incubation, while the bacteria removed ca 95% of both fats. Growth rate, biomass production and biomass yield per unit of fat consumed were all higher for the yeast compared to the bacterial consortium. The bacterial consortium exhibited autolysis and a significant decrease in its DCW value at the late growth phases of both fat substrate cultures. The main fatty acids (FAs) present in both fats were linoleic (Δ9,12C18:2), oleic (Δ9C18:1), palmitic (C16:0), palmitoleic (Δ9C16:1) and stearic (C18:0) acid. Both the bacterial consortium and Y. lipolytica preferentially removed Δ9C18:1 from the medium, while a negative selectivity against C18:0 was reported. Both inocula produced microbial mass that contained intra-cellular lipid quantities, but the bacterial consortium gave significantly higher lipid in DCW values compared with the yeast (maximum values up to ca 63% w/w for the butter and ca 42% w/w for the olive oil while the respective values for both lipids were 22%±2% w/w for Y. lipolytica). In all cases, intra-cellular lipids in DCW values decreased during the late growth phases, while their FA composition differed with those of the substrate fat

Lipid production by yeasts growing on commercial xylose in submerged cultures with process water being partially replaced by olive mill wastewaters

Six yeast strains belonging to Rhodosporidium toruloides, Lipomyces starkeyi, Rhodotorula glutinis and Cryptococcus curvatus were shake-flask cultured on xylose (initial sugar – S0 =70±10 g/L) under nitrogen-limited conditions. C. curvatus ATCC 20509 and L. starkeyi DSM 70296 were further cultured in media where process waters were partially replaced by the phenol-containing olive-mill wastewaters (OMWs). In flasks with S0≈100 g/L and OMWs added yielding to initial phenolic compounds concentration (PCC0) between 0.0 g/L (blank experiment) and 2.0 g/L, C. curvatus presented maximum total dry cell weight - TDCWmax ≈27 g/L, in all cases. The more the PCC0 increased, the fewer lipids were produced. In OMW-enriched media with PCC0≈1.2 g/L, TDCW=20.9 g/L containing ≈40% w/w of lipids was recorded. In L. starkeyi cultures, when PCC0≈2.0 g/L, TDCW≈25 g/L was synthesized, whereas lipids in TDCW =24-28% w/w, similar to the experiments without OMWs, were recorded. Non-negligible dephenolization and species-dependant decolorization of the wastewater occurred. A batch-bioreactor trial by C. curvatus only with xylose (S0≈110 g/L) was performed, and TDCW=35.1 g/L (lipids in TDCW=41.3% w/w) was produced. Yeast total lipids were composed of oleic and palmitic and to lesser extent linoleic and stearic acids. C. curvatus lipids were mainly composed of non-polar fractions (i.e. triacylglycerols)

Valorisation of waste bread for the production of yeast biomass by yarrowia lipolytica bioreactor fermentation

The increase in the wastage of bread, representing 12.5 million tons per year, causes ecological problems, such as the production of methane and CO2, when that waste bread (WB) is improperly managed. To reduce this ecological footprint, a more sustainable system of WB management must be set up. Based on its chemical composition, WB has a high potential to be used as feedstock for microbial growth and conversion into value-added bio products. The microbial valorisation of WB is a novel biotechnological approach to upgrading a waste into a renewable feedstock for bio-based industry, thus favouring the circular economy concept. Based on this, the aim of this study was to test WB as a feedstock for biomass production by Yarrowia lipolytica, which can be considered as a promising supplement for animal and human dietary products. The enzymatic hydrolysis of WB was primarily optimized for large-scale production in a bioreactor. The biomass production of Y. lipolytica strain K57 on WB hydrolysate-based media in batch bioreactor culture was then investigated. As a result, a very high starch to glucose conversion yield of 97% was obtained throughout optimised hydrolysis. At the end of 47 h of batch culture, a biomass higher than 62 g/L, specific growth rate of 0.37 h−1 and biomass yield of 0.45 g/g were achieved from a WB hydrolysate. Therefore, this study demonstrates that WB hydrolysate has a promising potential to be used as a feedstock for biomass production by Y. lipolytica strain K57 for food and animal diet applications.info:eu-repo/semantics/publishedVersio

Development of a circular oriented bioprocess for microbial oil production using diversified mixed confectionery side-streams

Diversified mixed confectionery waste streams were utilized in a two-stage bioprocess to formulate a nutrient-rich fermentation media for microbial oil production. Solid-state fermentation was conducted for the production of crude enzyme consortia to be subsequently applied in hydrolytic reactions to break down starch, disaccharides, and proteins into monosaccharides, amino acids, and peptides. Crude hydrolysates were evaluated in bioconversion processes using the red yeast Rhodosporidium toruloides DSM 4444 both in batch and fed-batch mode. Under nitrogen-limiting conditions, during fed-batch cultures, the concentration of microbial lipids reached 16.6−17 g·L−1 with the intracellular content being more than 40% (w/w) in both hydrolysates applied. R. toruloides was able to metabolize mixed carbon sources without catabolite repression. The fatty acid profile of the produced lipids was altered based on the substrate employed in the bioconversion process. Microbial lipids were rich in polyunsaturated fatty acids, with oleic acid being the major fatty acid (61.7%, w/w). This study showed that mixed food side-streams could be valorized for the production of microbial oil with high unsaturation degree, pointing towards the potential to produce tailor-made lipids for specific food applications. Likewise, the proposed process conforms unequivocally to the principles of the circular economy, as the entire quantity of confectionery by-products are implemented to generate added-value compounds that will find applications in the same original industry, thus closing the loop

Valorisation of side streams from wheat milling and confectionery industries for consolidated production and extraction of microbial lipids

Crude enzymes produced via solid state fermentation (SSF) using wheat milling by-products have been employed for both fermentation media production using flour-rich waste (FRW) streams and lysis of Rhodosporidium toruloides yeast cells. Filter sterilization of crude hydrolysates was more beneficial than heat sterilization regarding yeast growth and microbial oil production. The initial carbon to free amino nitrogen ratio of crude hydrolysates was optimized (80.2 g/g) in fed-batch cultures of R. toruloides leading to a total dry weight of 61.2 g/L with microbial oil content of 61.8 % (w/w). Employing a feeding strategy where the glucose concentration was maintained in the range of 12.2 – 17.6 g/L led to the highest productivity (0.32 g/L∙h). The crude enzymes produced by SSF were utilised for yeast cell treatment leading to simultaneous release of around 80% of total lipids in the broth and production of a hydrolysate suitable as yeast extract replacement

Rhodosporidium toruloides cultivated in NaCl-enriched glucose-based media: adaptation dynamics and lipid production

In the present report and for the first time in the international literature, the impact of the addition of NaCl upon growth and lipid production on the oleaginous yeast Rhodosporidium toruloides was studied. Moreover, equally for first time, lipid production by R. toruloides was performed under non-aseptic conditions. Therefore, the potentiality of R. toruloides DSM 4444 to produce lipid in media containing several initial concentrations of NaCl with glucose employed as carbon source was studied. Preliminary batch-flask trials with increasing amounts of NaCl revealed the tolerance of the strain against NaCl content up to 6.0% (w/v). However, 4.0% (w/v) of NaCl stimulated lipid accumulation for this strain, by enhancing lipid production up to 71.3% (w/w) per dry cell weight. The same amount of NaCl was employed in pasteurized batch-flask cultures in order to investigate the role of the salt as bacterial inhibiting agent. The combination of NaCl and high glucose concentrations was found to satisfactorily suppress bacterial contamination of R. toruloides cultures under these conditions. Batch-bioreactor trials of the yeast in the same media with high glucose content (up to 150 g/L) resulted in satisfactory substrate assimilation, with almost linear kinetic profile for lipid production, regardless of the initial glucose concentration imposed. Finally, fed-batch bioreactor cultures led to the production of 37.2 g/L of biomass, accompanied by 64.5% (w/w) of lipid yield. Lipid yield per unit of glucose consumed received the very satisfactory value of 0.21 g/g, a value amongst the highest ones in the literature. The yeast lipid produced contained mainly oleic acid and to lesser extent palmitic and stearic acids, thus constituting a perfect starting material for “second generation” biodiese

Industrial derivative of tallow: a promising renewable substrate for microbial lipid, single-cell protein and lipase production by Yarrowia lipolytica

The aim of the present study was to assess the potential of valorisation of a solid industrial derivative of tallow, composed of saturated free-fatty acids ("stearin"), by fermentations carried out by the yeast Yarrowia lipolytica ACA-DC 50109 in order to produce microbial lipid, biomass and extra-cellular lipase. High quantities of biomass were produced (biomass yield of around 1.1 \ub1 0.1 g of total biomass produced per g of fat consumed) when the organism was grown in shake flasks, regardless of the concentration of extra-cellular nitrogen present. Cellular lipids accumulated in notable quantities regardless of the nitrogen availability of the medium, though this process was clearly favoured at high initial fat and low initial nitrogen concentrations. The maximum quantity of fat produced was 7.9 mg/ml corresponding to 52.0% (wt/wt) of lipid in the dry biomass. Lipase production was critically affected by the medium composition and its concentration clearly increased with increasing concentrations of fat and extra-cellular nitrogen concentration reaching a maximum level of 2.50 IU/ml. Lipase concentration decreased in the stationary growth phase. In bioreactor trials, in which higher agitation and aeration conditions were employed compared with the equivalent trial in the flasks, significantly higher quantities of biomass were produced (maximum concentration 30.5 mg/ml, yield of 1.6 g of total biomass produced per g of fat consumed) while remarkably lower quantities of cellular lipids and extra-cellular lipase were synthesised. Numerical models successfully simulated both conversion of substrate fat into biomass and production and subsequent hydrolysis of extra-cellular lipase and presented a satisfactory predictive ability verifying the potential for single-cell protein and lipase production by Yarrowia lipolytica ACA-DC 50109. In all cultures, the mycelial form of the culture was dominant with few single cells present

Bioprocess development for biolubricant production using microbial oil derived via fermentation from confectionery industry waste

Microbial oil produced from confectionery and wheat milling side streams has been evaluated as novel feedstock for biolubricant production. Nutrient-rich fermentation media were produced by a two-step bioprocess involving crude enzyme production by solid state fermentation followed by enzymatic hydrolysis of confectionery industry waste. Among 5 yeast strains and 2 fungal strains cultivated on the crude hydrolysate, Rhodosporidium toruloides and Cryptococcus curvatus were selected for further evaluation for biolubricant production based on fermentation efficiency and fatty acid composition. The extracted microbial oils were enzymatically hydrolysed and the free fatty acids were esterified by Lipomod 34-MDP in a solvent-free system with trimethylolpropane (TMP) and neopentyl glycol (NPG). The highest conversion yields were 88% and 82.7% for NPG esters of R. toruloides and C. curvatus, respectively. This study also demonstrates that NPG esters produced from microbial oil have promising physicochemical properties for bio-based lubricant formulations that could substitute for conventional lubricants

Effect of impurities in biodiesel-derived waste glycerol on the performance and feasibility of biotechnological processes

The rapid development of biodiesel production technology has led to the generation of tremendous quantities of glycerol wastes, as the main by-product of the process. Stoichiometrically, it has been calculated that for every 100 kg of biodiesel, 10 kg of glycerol are produced. Based on the technology imposed by various biodiesel plants, glycerol wastes may contain numerous kinds of impurities such as methanol, salts, soaps, heavy metals and residual fatty acids. This fact often renders biodiesel-derived glycerol unprofitable for further purification. Therefore, the utilization of crude glycerol though biotechnological means represents a promising alternative for the effective management of this industrial waste. This review summarizes the effect of various impurities-contaminants that are found in biodiesel-derived crude glycerol upon its conversion by microbial strains in biotechnological processes. Insights are given concerning the technologies that are currently applied in biodiesel production, with emphasis to the impurities that are added in the composition of crude glycerol, through each step of the production process. Moreover, extensive discussion is made in relation with the impact of the nature of impurities upon the performances of prokaryotic and eukaryotic microorganisms, during crude glycerol bioconversions into a variety of high added-value metabolic products. Finally, aspects concerning ways of crude glycerol treatment for the removal of inhibitory contaminants as reported in the literature are given and comprehensively discusse