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- Integrated perspective on microbe-based production of itaconic acid: from metabolic and strain engineering to upstream and downstream strategiesPublication . Nascimento, Miguel Figueiredo; Marques, Nuno; Correia, Joana; Faria, Nuno Torres; Mira, Nuno P.; Ferreira, Frederico CasteloABSTRACT: The discovery of itaconic acid as a product of citric acid pyrolytic distillation in 1837 opened the possibility of using it as a polymer building block. Itaconic acid, featuring two carboxylic acids and an unsaturated group, can potentially be used as a building block in several chemical syntheses, with a particular emphasis on polymer manufacture. The elucidation of biochemical pathways originating from itaconic acid, first in Aspergillus terreus and, recently, in several species of the Ustilago genus, has intensified and diversified research focused on microbe-based itaconic acid production, including at an industrial scale. These efforts include the engineering of naturally producing species/strains along with the exploration of other species that do not naturally produce itaconic acid but may offer potential benefits. The use of renewable wastes or sugar-enriched residues as substrates to produce itaconic acid, from a circular bioeconomy perspective, is another important aspect of the advancements in microbial itaconic acid production. This review provides an overview of the achievements as well as the challenges concerning the engineering of the producing strains/species, substrate selection, optimisation of bioreactor operation, and downstream itaconic acid purification methods.
- Conversion of cellulosic materials into glycolipid biosurfactants, mannosylerythritol lipids, by Pseudozyma spp. under SHF and SSF processesPublication . Faria, Nuno Torres; Santos, Marisa V.; Ferreira, Carla; Marques, Susana; Ferreira, Frederico Castelo; Fonseca, CésarBackground: Mannosylerythritol lipids (MEL) are glycolipids with unique biosurfactant properties and are produced by Pseudozyma spp. from different substrates, preferably vegetable oils, but also sugars, glycerol or hydrocarbons. However, solvent intensive downstream processing and the relatively high prices of raw materials currently used for MEL production are drawbacks in its sustainable commercial deployment. The present work aims to demonstrate MEL production from cellulosic materials and investigate the requirements and consequences of combining commercial cellulolytic enzymes and Pseudozyma spp. under separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) processes. Results: MEL was produced from cellulosic substrates, Avicel® as reference (>99% cellulose) and hydrothermally pretreated wheat straw, using commercial cellulolytic enzymes (Celluclast 1.5 L® and Novozyme 188®) and Pseudozyma antarctica PYCC 5048T or Pseudozyma aphidis PYCC 5535T. The strategies included SHF, SSF and fed-batch SSF with pre-hydrolysis. While SSF was isothermal at 28°C, in SHF and fed-batch SSF, yeast fermentation was preceded by an enzymatic (pre-)hydrolysis step at 50°C for 48 h. Pseudozyma antarctica showed the highest MEL yields from both cellulosic substrates, reaching titres of 4.0 and 1.4 g/l by SHF of Avicel® and wheat straw (40 g/l glucan), respectively, using enzymes at low dosage (3.6 and 8.5 FPU/gglucan at 28°C and 50°C, respectively) with prior dialysis. Higher MEL titres were obtained by fed-batch SSF with pre-hydrolysis, reaching 4.5 and 2.5 g/l from Avicel® and wheat straw (80 g/l glucan), respectively. Conclusions: This work reports for the first time MEL production from cellulosic materials. The process was successfully performed through SHF, SSF or Fed-batch SSF, requiring, for maximal performance, dialysed commercial cellulolytic enzymes. The use of inexpensive lignocellulosic substrates associated to straightforward downstream processing from sugary broths is expected to have a great impact in the economy of MEL production for the biosurfactant market, inasmuch as low enzyme dosage is sufficient for good systems performance.
- Moesziomyces spp. cultivation using cheese whey: new yeast extract-free media, beta-galactosidase biosynthesis and mannosylerythritol lipids productionPublication . Nascimento, Miguel Figueiredo; Barreiros, Ricardo; Oliveira, Ana Cristina; Ferreira, Frederico Castelo; Faria, Nuno TorresABSTRACT: Mannosylerythritol lipids (MELs) are biosurfactants with excellent biochemical properties and a wide range of potential applications. However, high production costs, low productivity and unsatisfactory scale-up production have hampered commercial adoption. Herein, we report for the first time the beta-galactosidase production by Moesziomyces spp. from different sugars (D-galactose, D-glucose and D-lactose), with D-galactose being the best beta-galactosidase inducer, with 11.2 and 63.1 IU/mg(biomass), for Moesziomyces aphidis 5535(T) and Moesziomyces antarcticus 5048(T), respectively. The production of this enzyme allows to break down D-lactose and thus to produce MEL directly from D-lactose or cheese whey (a cheese industry by-product). Remarkably, when CW was used as sole media component (carbon and mineral source), in combination with waste frying oil, MEL productivities were very close (1.40 and 1.31 g(MEL)/L/day) to the ones obtained with optimized medium containing yeast extract (1.92 and 1.50 g(MEL)/g(susbtrate)), both for M. antarcticus and M. aphidis. The low-cost, facile and efficient process which generates large amounts of MELs potentiates its industrialization.
- Production of Mannosylerythritol Lipids Using Oils from Oleaginous Microalgae: Two Sequential Microorganism Culture ApproachPublication . Nascimento, Miguel Figueiredo; Coelho, Tiago; Reis, Alberto; Gouveia, Luisa; Faria, Nuno Torres; Ferreira, Frederico CasteloABSTRACT: Mannosylerythritol lipids (MELs) are biosurfactants with excellent biochemical properties and a wide range of potential applications. However, most of the studies focusing on MELs high titre production have been relying in the use of vegetable oils with impact on the sustainability and process economy. Herein, we report for the first time MELs production using oils produced from microalgae. The bio-oil was extracted from Neochloris oleoabundans and evaluated for their use as sole carbon source or in a co-substrate strategy, using as an additional carbon source D-glucose, on Moesziomyces spp. cultures to support cell growth and induce the production of MELs. Both Moesziomyces antarcticus and M. aphidis were able to grow and produce MELs using algae-derived bio-oils as a carbon source. Using a medium containing as carbon sources 40 g/L of D-glucose and 20 g/L of bio-oils, Moesziomyces antarcticus and M. aphidis produced 12.47 +/- 0.28 and 5.72 +/- 2.32 g/L of MELs, respectively. Interestingly, there are no significant differences in productivity when using oils from microalgae or vegetable oils as carbon sources. The MELs productivities achieved were 1.78 +/- 0.04 and 1.99 +/- 0.12 g/L/h, respectively, for M. antarcticus fed with algae-derived or vegetable oils. These results open new perspectives for the production of MELs in systems combining different microorganisms.
- Production of glycolipid biosurfactants, mannosylerythritol lipids, from pentoses and D-glucose/D-xylose mixtures by Pseudozyma yeast strainsPublication . Faria, Nuno Torres; Santos, Marisa V.; Fernandes, Pedro; Fonseca, Luís Lopes; Fonseca, César; Ferreira, Frederico CasteloThe pentose-assimilating capacity of yeasts from the genus Pseudozyma, P. antarctica PYCC 5048T, P.aphidis PYCC 5535Tand P. rugulosa PYCC 5537T, was exploited towards the production of mannosylery-thritol lipids (MEL), a glycolipid with biosurfactant properties. The three strains tested were able togrow on d-xylose and l-arabinose with similar maximum specific growth rates to those estimated on d-glucose (around 0.2 h-1). The highest MEL titres (4.8–5.4 g/l) and yields (0.11–0.14 g/g) from d-xylosewere found in P. antarctica PYCC 5048T, which presented similar values to those estimated on D-glucose and on D-xylose/D-glucose mixtures. P. rugulosa PYCC 5537T showed a pattern of sugar conversion into MEL similar to P. antarctica, but at 40% lower titres. P. aphidis PYCC 5535T presented lower MEL titres from D-xylose (1.2 g/l) than from D-glucose (3.4 g/l). Nitrate supply increased sugar consumption rate and, when accompanied by D-glucose or D-xylose feeding, additional biomass production. In this case, sugar was completely consumed before sugar feeding at day 7, but not when feeding is performed at day 4. Higher MEL titres where obtained for the later condition reaching 7.3 g/l and 5.8 g/l, in fed-atchcultures with glucose and xylose, respectively.
- Monitoring Yeast Cultures Grown on Corn Stover Hydrolysate for Lipid ProductionPublication . Fontes, Afonso; Francisco, Ricardo; Ferreira, Frederico Castelo; Faria, Nuno Torres; Marques, Susana; Reis, Alberto; Moura, Patrícia; Lukasik, Rafal M.; Santos, José A. L.; Silva, Teresa Lopes daABSTRACT: Microbial oils can be used as an alternative sustainable and renewable feedstock to fossil reserves for producing lubricants and polyurethane materials. Two oleaginous yeasts were grown on non-detoxified corn stover hydrolysate supplemented with corn steep liquor and mineral medium in shake flasks. Trichosporon oleaginosus DSM 11815 displayed the highest lipid production. This strain was further cultivated in a bench bioreactor, using the same culture medium, under a batch regime. Flow cytometry was used to monitor the T. oleaginosus culture using the dual staining technique (SYBR Green and PI) for cell membrane integrity detection. Values of 42.28% (w/w) and 0.06 g/Lh lipid content and lipid productivity, respectively, were recorded for T. oleaginosus cultivated in the bench bioreactor operated under a batch regime. During the cultivation, most of the yeast cells maintained their integrity. T. oleaginosus has the potential to be used as an oil microbial source for a wide range of industrial applications. In addition, it is robust in adverse conditions such as lignocellulosic hydrolysate exposure and oxygen-limiting conditions. Flow cytometry is a powerful and useful tool for monitoring yeast cultivations on lignocellulosic hydrolysates for cell count, size, granularity, and membrane integrity detection.
- High cellulase-free xylanases production by Moesziomyces aphidis using low-cost carbon and nitrogen sourcesPublication . Faria, Nuno Torres; Marques, Susana; Cerejo, Joana; Vorobieva, Ekaterina; Fonseca, CésarABSTRACT: Background Enzymes involved in xylan hydrolysis have several industrial applications. Selection of efficient microbial hosts and scalable bioreaction operations can lower enzyme production costs and contribute to their commercial deployment. This work aims at investigating the Moesziomyces aphidis yeast cultivation conditions that deliver maximal xylanase titres, yields and productivities using low-cost nitrogen (N) and carbon (C) sources. Results NaNO3 and KNO3 supplementation improved xylanase production 2.9- and 2.7-fold (against 67.2 U mL(-1)), respectively, using xylan as C source. Interestingly, the use of KNO3, instead of NaNO3, results in 2- to 3-fold higher specific activity, highlighting the potassium ion role. In addition, this study investigates synergetic effects on using ionic and organic N sources. A 4.9-fold increase in xylanase production, with high specific activity, is attained combining KNO3 and corn steep liquor (CSL). Exploring the previous findings, this study reports one of the highest extracellular xylanase production titres (864.7 U mL(-1)) by yeasts, using a media formulation containing dilute-acid pre-treated brewery spent grains (BSG), as C source and inducer, supplemented with KNO3 and CSL. Replacement of dilute-acid pre-treatmed BSG by untreated BSG had low impact on xylanase production, of only 6%. Conclusion Efficient production of M. aphidis xylanolytic enzymes, using low-cost N and C sources, is attractive for deployment of on-site enzyme production targeting different biotechnological applications under circular economy and biorefinery concepts. Potential xylanases end-users include industries such as brewing (using BSG as substrate for enzyme production), pulp and paper (benefiting from the cellulase-free xylanase activity) or lignocellulosic ethanol (for cellulase supplementation).
- Production of sustainable aviation fuel precursors using the oleaginous yeast Rhodotorula toruloides PYCC 5615 cultivated on eucalyptus bark hydrolysatePublication . Saraiva Lopes da Silva, Maria Teresa; Dutra, Francisca; Gomes, Miguel; Costa, Paula; Paradela, Filipe; Ferreira, Frederico Castelo; Torres Faria, Nuno Ricardo; Mugica, Paula; Pinheiro, Helena M.; Sá-Correia, Isabel; Gírio, Francisco; Marques, SusanaABSTRACT: Sustainable aviation fuels (SAF) obtained from renewable sources of carbon can reduce carbon dioxide emissions and contribute for mitigating climate changes. In the present study, the yeast Rhodotorula toruloides PYCC 5615 was found to be highly promising for the bioconversion of eucalyptus bark hydrolysate and the accumulation of intracellular lipids which were further thermochemically processed to bioenergy intermediaries for SAF production. Two growth medium formulations were tested. Eucalyptus bark hydrolysate, obtained by steam explosion followed by enzymatic hydrolysis, was supplemented with yeast nitrogen base medium or with corn steep liquor and mineral medium. The latter produced the highest fatty acid content and productivity (30 % w/w and 0.11 g/ (L.h) respectively). Thereafter, the whole yeast biomass (WB) and the de-oiled biomass (DOB), obtained after lipid extraction, were processed into Bio-crude using a hydrothermal liquefaction (HTL) reactor, with a yield of approximate to 40 % (w/w). The two obtained Bio-crude fractions and the yeast lipids fraction (YL) were further upgraded by hydrodeoxygenation (HDO), to remove oxygen atoms and increase the hydrocarbon content, resulting in a Bio-crude composed of linear long-chain fatty acids suitable for processing to SAF. The best Bio-crude characteristics was observed for WB and YL fractions, with 34.8 % and 40.7 % of hydrocarbons, respectively. Both WB and YL hydrocarbons were composed of C15-C17 compounds. These results demonstrate the potential of an integrated process based on microbial oils from R. toruloides PYCC 5615 to produce SAF precursors from Eucalyptus bark residues, contributing for the sustainable jetfuel bioproduction process.
- Direct xylan conversion into glycolipid biosurfactants,mannosylerythritol lipids, by Pseudozyma antarctica PYCC 5048TPublication . Faria, Nuno Torres; Marques, Susana; Fonseca, César; Ferreira, Frederico CasteloMannosylerythritol lipids (MEL) are glycolipid biosurfactants, produced by Pseudozyma spp., with increasing commercial interest. While MEL can be produced from d-glucose and d-xylose, the direct conversion of the respective lignocellulosic polysaccharides, cellulose and xylan, was not reported yet. The ability of Pseudozyma antarctica PYCC 5048T and Pseudozyma aphidis PYCC 5535T to use cellulose (Avicel®) and xylan (beechwood) as carbon and energy source has been assessed along with their capacity of producing cellulolytic and hemicellulolytic enzymes, toward a consolidated bioprocess (CBP) for MEL production. The yeasts assessed were neither able to grow in medium containing Avicel® nor produce cellulolytic enzymes under the conditions tested. On contrary, both yeasts were able to efficiently grow in xylan, but MEL production was only detected in P. antarctica PYCC 5048T cultures. MEL titers reached 1.3 g/l after 10 days in batch cultures with 40 g/l xylan, and 2.0 g/l in fed-batch cultures with xylan feeding (additional 40 g/l) at day 4. High levels of xylanase activities were detected in xylan cultures, reaching 47–62 U/ml (31–32 U/mg) at 50 °C, and still exhibiting more than 10 U/ml under physiological temperature (28 °C). Total ß-xylosidase activities, displayed mainly as wall-bounded and extracellular activity, accounted for 0.154 and 0.176 U/ml in P. antarctica PYCC 5048T and P. aphidis PYCC 5535T cultures, respectively. The present results demonstrate the potential of Pseudozyma spp. for using directly a fraction of lignocellulosic biomass, xylan, and combining in the same bioprocess the production of xylanolytic enzymes with MEL production.