Teresa Lopes da Silva

Crypthecodinium cohnii with emphasis on DHA production: a review

Docosahexaenoic acid (DHA) is a polyunsaturated fatty acid (PUFA) that belongs to the ω-3 group. In recent years, DHA has attracted much attention because of its recognized beneficial effect on human health. At present, fish oil is the major source of DHA, but it may be produced by microorganisms with additional benefits. Marine microorganisms may contain large amounts of DHA and are considered a potential source of this important fatty acid. Some of these organisms can be grown heterotrophically on organic substrates without light, offering the possibility of greatly increasing microalgal cell concentration under controlled and monitored conditions, resulting in a very high quality product. Among the heterotrophic marine dinoflagellates, Crypthecodinium cohnii has been identified as a prolific producer of DHA. The organism is extraordinary in that it produces no other PUFAs than DHA in its cell lipid in any significant amount, which makes the DHA purification process very attractive, particularly for pharmaceutical and nutraceutical applications. This paper reviews recent advances in the biotechnological production of DHA by C. cohnii.

Supercritical fluid extraction of lipids from the heterotrophic microalga Crypthecodinium cohnii

Microalgae biomass can be a feasible source of ω‐3 fatty acids due to its stable and reliable composition. In the present study, the Crypthecodinium cohnii growth and docosahexaenoic acid (DHA, 22:6ω3) production in a 100 L glucose‐fed batch fermentation was evaluated. The lipid compounds were extracted by supercritical carbon dioxide (SC‐CO2) from C. cohnii CCMP 316 biomas, was and their fatty acid composition was analysed. Supercritical fluid extraction runs were performed at temperatures of 313 and 323 K and pressures of 20.0, 25.0 and 30.0 MPa. The optimum extraction conditions were found to be 30.0 MPa and 323 K. Under those conditions, almost 50% of the total oil contained in the raw material was extracted after 3 h and the DHA composition attained 72% w/w of total fatty acids. The high DHA percentage of total fatty acids obtained by SC‐CO2 suggested that this extraction method may be suitable for the production of C. cohnii value added products directed towards pharmaceutical purposes. Furthermore, the fatty acid composition of the remaining lipid fraction from the residual biomass with lower content in polyunsaturated fatty acids could be adequate for further uses as feedstock for biodiesel, contributing to the economy of the overall process suggesting an integrated biorefinery approach.

Integrated microbial processes for biofuels and high value-added products: the way to improve the cost effectiveness of biofuel production

The production of microbial biofuels is currently under investigation, as they are alternative sources to fossil fuels, which are diminishing and their use has a negative impact on the environment. However, so far, biofuels derived from microbes are not economically competitive. One way to overcome this bottleneck is the use of microorganisms to transform substrates into biofuels and high value-added products, and simultaneously taking advantage of the various microbial biomass components to produce other products of interest, as an integrated process. In this way, it is possible to maximize the economic value of the whole process, with the desired reduction of the waste streams produced. It is expected that this integrated system makes the biofuel production economically sustainable and competitive in the near future. This review describes the investigation on integrated microbial processes (based on bacteria, yeast, and microalgal cultivations) that have been experimentally developed, highlighting the importance of this approach as a way to optimize microbial biofuel production process.

Applications and perspectives of multi-parameter flow cytometry to microbial biofuels production processes

Conventional microbiology methods used to monitor microbial biofuels production are based on off-line analyses. The analyses are, unfortunately, insufficient for bioprocess optimization. Real time process control strategies, such as flow cytometry (FC), can be used to monitor bioprocess development (at-line) by providing single cell information that improves process model formulation and validation. This paper reviews the current uses and potential applications of FC in biodiesel, bioethanol, biomethane, biohydrogen and fuel cell processes. By highlighting the inherent accuracy and robustness of the technique for a range of biofuel processing parameters, more robust monitoring and control may be implemented to enhance process efficiency.

Selecting low-cost carbon sources for carotenoid and lipid production by the pink yeast Rhodosporidium toruloides NCYC 921 using flow cytometry

The present work studied low-cost carbon sources for carotenoid and lipid production using the yeast Rhodosporidum toruloides NCYC 921. Carob pulp syrup and sugarcane molasses at different concentrations were used as low-cost carbon sources in R. toruloides batch cultivations. Carob pulp syrup containing a total sugar concentration of 75 g L(-1) induced the highest total fatty acid productivity (1.90 g L(-1)h(-1)) and the highest carotenoid productivity (9.79 μg L(-1)h(-1)). Flow cytometric analysis revealed that most of the yeast cells (>60%) grown on carob pulp syrup displayed intact polarised membranes, conversely to the cells grown on sugarcane molasses, wherein a large proportion (>45%) displayed permeabilised cytoplasmic membranes.

Using multi-parameter flow cytometry to monitor the yeast Rhodotorula glutinis CCMI 145 batch growth and oil production towards biodiesel.

Multi-parameter flow cytometry was used to monitor cell intrinsic light scatter, viability, and lipid content of Rhodotorula glutinis CCMI 145 cells grown in shake flasks. Changes in the side light scatter and forward light scatter were detected during the yeast batch growth, which were attributed to the different yeast growth phases. A progressive increase in the proportion of cells stained with PI (cells with permeabilized cytoplasmic membrane) was observed during the yeast growth, attaining 79% at the end of the fermentation. A high correlation between the Nile Red fluorescence intensity measured by flow cytometry and total lipid content assayed by the traditional gravimetric lipid analysis was found for this yeast, making this method a suitable and quick technique for the screening of yeast strains for lipid production and optimization of biofuel production bioprocesses. Medium growth optimization for enhancement of the yeast oil production is now in progress.

New dual-stage pH control fed-batch cultivation strategy for the improvement of lipids and carotenoids production by the red yeast Rhodosporidium toruloides NCYC 921

The optimal medium pH to produce biomass and fatty acids by the red yeast Rhodosporidium toruloides NCYC 921 is 4.0, and to produce carotenoids is 5.0. Based on this difference, a dual-stage pH control fed-batch cultivation strategy for the enhancement of lipids and carotenoids production by this yeast was studied. The results showed that when the yeast growth phase was conducted at pH 4.0, and the products accumulation phase was conducted at pH 5.0, biomass, total fatty acid and total carotenoid productivities were significantly improved comparing with the yeast fed batch cultivations carried out at fixed medium pH (4 or 5). Under dual-stage pH control conditions, the biomass, carotenoids and lipids productivities attained 2.35 g/Lh, 0.29 g/Lh and 0.40 g/Lh, respectively. It was also observed that the oxygen played a major role in the yeast carotenoid production.

Monitoring Rhodotorula glutinis CCMI 145 physiological response and oil production growing on xylose and glucose using multi-parameter flow cytometry.

Flow cytometry was used to monitor the lipid content, viability and intrinsic light scatter properties of Rhodotorula glutinis CCMI 145 cells growing on batch cultures using xylose and glucose as carbon sources. The highest lipid content was observed for cells grown on glucose, at the end of the exponential phase (17.8% w/w). The proportion of cells stained with PI attaining 77% at the end of the glucose growth. Cells growing on xylose produced a maximum lipid content of 10.6% (w/w), at the stationary phase. An increase in the proportion of cells stained with PI was observed, reaching 29% at the end of xylose growth. Changes in the side and forward light scatter detected during the yeast batch cultures supported that R. glutinis cells grown on glucose experienced harsher conditions, resulting in a high level of cytoplasmic membrane damage, which did not occur when R. glutinis cells grew on xylose.

The use of multi-parameter flow cytometry to study the impact of n -dodecane additions to marine dinoflagellate microalga Crypthecodinium cohnii batch fermentations and DHA production

The physiological response of Crypthecodinium cohnii batch cultivations and docosahexaenoic acid (DHA) production to n-dodecane additions were studied. Different n-dodecane concentrations [0, 0.5, 1, 2.5, 5, 10 and 20% (v/v)] were added to preliminary shake flask cultivations. The n-dodecane fraction that gave best results in terms of biomass and DHA production was 0.5% (v/v). The n-dodecane fractions of 2.5, 5, 10 and 20% (v/v) to C. cohnii preliminary shake flask cultures inhibited the microalgal growth and DHA production, although a high proportion of cells with intact cytoplasmic membrane was present in the end of these fermentations. After the addition of a pulse of n-dodecane (0.5% v/v) to C. cohnii exponential growing cells in a bioreactor, glucose uptake volumetric rate increased 2.5-fold, while biomass production volumetric rate increased 2.8-fold. The specific growth rate was increased 1.5-fold. The DHA % in biomass, DHA % of TFA and DHA concentration also increased (54, 22 and 58%, respectively), after the n-dodecane addition. At this n-dodecane fraction (0.5% v/v), multi-parameter flow cytometry demonstrated that C. cohnii cell membrane integrity was not affected. The results demonstrated that the addition of 0.5% of n-dodecane (v/v) to C. cohnii fermentations can be an easy and cheap way for enhancing the biomass and DHA production, avoiding the use of high speed rates (resulting in important power agitation costs) that affects the microalga proliferation and increases the bioprocess costs. A new strategy to improve the DHA production from this microalga in two-phase large-scale bioreactors is now in progress.

Effects of hydrocarbon additions on gas-liquid mass transfer coefficients in biphasic bioreactors

The effects of aliphatic hydrocarbons (n-hexadecane andn-dodecane) on the volumetric oxygen mass transfer coefficient (kLa) were studied in flat alveolar airlift reactor and continuous stirred tank reactors (CSTRs). In the flat alveolar airlift reactor, high aeration rates (>2 vvm) were required in order to obtain efficient organic-aqueous phase dispersion and reliablekLa measurements. Addition of 1% (v/v)n-hexadecane orn-dodecane increased thekla 1.55-and 1.33-fold, respectively, compared to the control (superficial velocity: 25.8×10−3 m/s, sparger orifice diameter: 0.5 mm). Analysis of the gas-liquid interfacial areaa and the liquid film mass transfer coefficientkL suggests that the observedkLa increase was a function of the medias liquid film mass transfer. Addition of 1% (v/v)n-hexadecane orn-dodecane to analogous setups using CSTRs led to akLa increase by a factor of 1.68 and 1.36, respectively (superficial velocity: 2.1×10−3 m/s, stirring rate: 250 rpm). These results propose that low-concentration addition of oxygen-vectors to aerobic microbial cultures has additional benefit relative to incubation in purely aqueous media.