Lard as a suitable and cheap substrate for microbial lipids production by Yarrowia lipolytica

[Excerpt] Current agricultural and industrial practices have led to the generation of large amounts of various low-value or negative cost crude wastes, which are difficult and economically notattractive to treat and valorize. One important example of waste generation is animal fat, commonly found in tanning process and slaughterhouses. These wastes, in which the lipids are often the main and most problematic components, are not currently used effectively and there are almost no application methods to recover the respective value. (...

Valorisation of wastes for single cell oil production by Yarrowia lipolytica

Various strains of oleaginous microorganisms, mainly fungi and yeast, have been widely used for the production of single cell oil (SCO) rich in polyunsaturated fatty acids or having an exceptional triacylglycerol structure. Difficulties for industrial scale production are related to the high cost of fermentation and oil extraction. The quantity of oil accumulated per unit of dry cellular mass is a critical factor that influences the final cost of SCO. The economics of these bioprocess become more favourable when zero or negative value waste substrates are utilized as carbon or nitrogen sources. Although utilisation of crude glycerol in the fermentation medium without prior purification offers a remarkable advantage against the traditional use of pure glycerol as substrate only few reports have appeared in the literature on the use of this substrate as sole carbon sources. The aim of current investigation was to assess the potentialities of valorisation of crude glycerol and lard, residues used as a carbon source by Yarrowia lipolytica strain in order to the production of SCO. Batch fermentations in 1-L Erlenmeyer flasks were performed using pure glycerol, crude glycerol and lard as carbon sources, with different concentrations (20 g/L, 50 g/L and 80 g/L). Y. lipolytica W29 was pre-grown overnight in YPD medium, centrifuged and resuspended in each carbon source medium, supplemented with yeast extract (0.5 g/L). The production of SCO was carried out during 168 h at 27 ºC and 185 rpm. Yeast cells were able to grown on all carbon sources, although a slight inhibition with 80 g/L of pure and crude glycerol was observed. No significant differences on final cell dry weight were noted between the carbon sources, reaching approximately 5 g/L. The increase in carbon source concentration leads to an improvement in lipid accumulation inside the cells. The highest amount of reserve lipid was observed in medium with lard 80 g/L (21.3 % of cell dry weight). The strain showed the tendency to degrade its storage lipids when grown on 20 g/L of each carbon sources, probably due to the early consumption of substrate. Crude glycerol batch fermentation at 50 g/L in a 2-L bioreactor led to an accumulation of lipid content inside the cells of 37 % cell dry weight. The results of this study suggest that SCO could be produced by Y. lipolytica W29 using low-cost substrates, such as crude glycerol and lard

Application of hyperbaric bioreactors for bioprocess development

For many years pressure was disregarded by biotechnologists when considering processes based on microbial cultures, due to the idea of incompatibility of life in pressure environments above atmospheric pressure. However, the discovery of microbial life in such environments has refuted this idea. Moreover, in industrial bioreactors microorganisms are exposed to spatial gradients of pressure (with average values above atmospheric pressure), since its value is a function of liquid height. Consequently, local differences of gas solubility occur in industrial bioreactors. This can have a strong impact on overall productivities, namely on bioreactor scale-up. Thus, simulations of these conditions in lab-scale reactors working at wide-ranging total pressure are of great importance. On the other hand, the use of hyperbaric bioreactors (working with total gas pressure of several units or tens above atmospheric pressure), can be applied for oxygen transfer rate improvement for aerobic microbial cultures [1]. In fact, the oxygen demand in high cell density cultivation exceeds by far the maximum oxygen transfer capacity of conventional bioreactors such as stirred tanks or bubble columns. Our group has been studying the effects of increase air pressure on microbial physiology in order to assess the limits of its use for oxygen unlimited cellular growth. This work is focused on the application of hyperbaric bioreactors for the cultivation of different yeast species of industrial interest, such as Yarrowia lipolytica and Pichia pastoris. Besides cellular growth enhancement by oxygen solubility increase, also effects on enzymes activities were found, either of metabolic pathways, oxidative stress cellular response mechanism and extracellular enzymes

Enhanced growth of Pichia pastoris under increased air pressure on different carbon sources

Pichia pastoris has many biotechnological applications. Two aspects of the species have contributed to its utility: (1) fermentation techniques were developed for maintaining extremely high cell densities in excess of 100 g/L dry weight, and (2) because P. pastoris assimilates methanol, the expression system is linked with alcohol oxidase, which is abundantly produced in the presence of methanol. The high oxygen demand of methanol metabolism and cultivation at very high-cell-density makes oxygen supply a major parameter in Pichia pastoris cultivation. Previous work demonstrated that hyperbaric air could be successfully applied to yeast cultivation, as a way of improving the oxygen transfer rate (OTR) to aerobic cultures [1]. In the present work, we investigate whether increasing air pressures may lead to increasing biomass yields of P. pastoris, growing with four carbon sources, without giving rise to unbalance oxidative stress. Pichia pastoris strain was grown in glucose, pure glycerol, crude glycerol from biodiesel industry and methanol media under total air pressure from 1 bar to 5 bar. In all the experiments, the cultures reached maximum cell density at 5 bar of total air pressure. A 4fold increase on specific growth rate was obtained at 5 bar on glycerol and crude glycerol compared to the value at atmospheric pressure. Biomass yield was also enhanced by air pressure rise, for all carbon sources. With 5 bar air pressure biomass yield (g cells/g carbon) was 0.97 and 1.86 whereas at 1 bar was 0.67 and 0.77, respectively in methanol and glycerol media

CBmeter- a new medical device for early screening of metabolic diseases

Type 2 diabetes mellitus (T2DM) is a highly prevalent disease worldwide which is asymptomatic in about 44% of patients being critical to search for new ways of early diagnosis. Recent studies have demonstrated that the etiology of this disease may be associated with alterations in the function of the carotid body (CB), a chemosensor organ located within the bifurcation of the carotid artery. In animal models of metabolic syndrome it was observed that the CBs are overactivated, underlying diseases such as obesity, hypertension and T2DM. This discovery provided a new paradigm in the neuroendocrinology field, suggesting that diagnostic function of the CBs has predictive value for the development of metabolic diseases. Despite this fact, it is not common in clinical practice to look at the CBs as organs associated with endocrine dysfunction and we believe this is probably due to the nonexistence of a user-friendly, portable medical device that diagnosis the function of the CBs.info:eu-repo/semantics/publishedVersio

Effect of increased air pressure in gluconic acid production by Aspergillus niger

Gluconic acid (GA) is an organic acid with many applications in food and chemical industries. GA is produced mainly by biotechnological processes and Aspergillus niger is the most used microorganism. This acid is obtained by the oxidation of glucose catalyzed by the enzyme glucose oxidase and this reaction has a high oxygen demand. Usually, oxygen is the major limiting factor of the process in common stirred tank bioreactors. This limitation can be overcome by increasing the total air pressure of the bioreactor. The goal of this work is the study of the improvement of oxygen mass transfer rate (OTR) from air to culture medium in the production of GA by A. niger 9213 using an hyperbaric bioreactor. Batch cultures were performed using glucose as substrate in the presence of calcium carbonate, in a stainless steel stirred tank bioreactor of 400 mL of working volume, at 1 vvm of aeration rate under 1 bar and 4 bar of total air pressure, corresponding to OTR values of 384 mg·L-1h-1 and 768 mg·L-1h-1, respectively. The increase of air pressure led to a significant improvement of GA production. A 5-fold increase on GA productivity was reached (around 3 g·L-1h-1) by the air pressure raise as well as on glucose uptake rate. The positive effect of pressure on GA production by A. niger was obtained using spores solution as inoculum or a pre-grown mycelium culture. The use of air pressure increase is an effective alternative way of OTR improvement and this showed its applicability for GA production. It presents several advantages compared to the use of oxygen-enriched air that is costly and requires special handling.This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2019 unit and the PhD grant SFRH/BD/129475/2017, and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte.info:eu-repo/semantics/publishedVersio

Use of pressurized and airlift bioreactors for citric acid production by Yarrowia lipolytica from crude glycerol

Citric acid production is generally carried out in an aqueous medium in stirred tank reactors (STR), where the solubility of oxygen is low and the oxygen demand of microbial cultures is high. Thus, for this bioprocess, providing adequate oxygen mass transfer rate (OTR) from the gas phase into the aqueous culture medium is the main challenge of bioreactor selection and operation. In this study, citric acid production by Yarrowia lipolytica W29 from crude glycerol, in batch cultures, was performed in two non-conventional bioreactors normally associated with high mass transfer efficiency: a pressurized STR and an airlift bioreactor. Increased OTR was obtained by raising the total air pressure in the pressurized STR and by increasing the aeration rate in the airlift bioreactor. An improvement of 40% in maximum citric acid titer was obtained by raising the air pressure from 1 bar to 2 bar, whereas, in the airlift bioreactor, a 30% improvement was attained by increasing the aeration rate from 1 vvm to 1.5 vvm. Both bioreactor types can be successfully applied for the citric acid production process using alternative ways of improving OTR than increasing mechanical stirring power input, thus leading to important operating saving costs.This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit; by LABBELS—Associate Laboratory in Biotechnology, Bioengineering and Microelectromechanical Systems, LA/P/0029/2020.info:eu-repo/semantics/publishedVersio

Valorisation of glycerol by Pichia pastoris under increased air pressure

Pichia pastoris has many biotechnological applications. Two aspects of the species have contributed to its utility: (1) fermentation techniques were developed for maintaining extremely high cell densities in excess of 100 g/L dry weight, and (2) because P. pastoris assimilates methanol, the expression system is linked with alcohol oxidase, which is abundantly produced in the presence of methanol. Glycerol is regularly used as the main initial carbon source in P. pastoris fermentations to increase cell concentration. The recent abundance of crude glycerol, coming out as the main byproduct of biodiesel production, made possible the use of crude glycerol as the carbon source for bioprocesses with the methylotrophic Pichia pastoris. In 2009, 276 biodiesel plants were operating in Europe. The rapidly expanding market for biodiesel is decreasing the cost and increasing the glycerol availability, as typical biodiesel production processes generate around 10 % (wt) glycerol of the total amount of biodiesel produced. Batch fermentations were performed in a hyperbaric reactor to study the effect of increasing total air pressure on Pichia pastoris CBS 2612 growing on pure glycerol, crude glycerol and methanol. In the experiments with pure glycerol, a total pressure of 5 bar led to a specific growth rate 3-fold higher than the obtained under 1 bar. Also with crude glycerol, a 4-fold improvement in specific growth rate was obtained in the experiment at 5 bar compared to the control trial. Biomass yield was also enhanced by air pressure rise, for all carbon sources. Under 5 bar biomass yield (mass of cells per mass of carbon) was 0.97, 1.86 and 3.65 whereas at 1 bar was 0.67, 1.72 and 0.9 respectively in methanol, pure glycerol and crude glycerol media. The current low cost of crude glycerol from the biodiesel production together with the present results shows the possibility of producing heterologous proteins using hyperbaric air with high productivity and at reduced costs