Lipid and Carotenoid Production by a Rhodosporidium toruloides and Tetradesmus obliquus Mixed Culture Using Primary Brewery Wastewater Supplemented with Sugarcane Molasses and Urea.

In this study, Rhodosporidium toruloides and Tetradesmus obliquus were used for lipid and carotenoid production in mixed cultures using primary brewery wastewater (PBWW) as a culture medium, supplemented with sugarcane molasses (SCM) as a carbon source and urea as a nitrogen source. To improve biomass, lipid, and carotenoid production by R. toruloides and T. obliquus mixed cultures, initial SCM concentrations ranging from 10 to 280 g L-1 were tested. The medium that allowed higher lipid content (26.2% w/w dry cell weight (DCW)) and higher carotenoid productivity (10.47 µg L-1 h-1) was the PBWW medium supplemented with 100 g L-1 of SCM and 2 g L-1 of urea, which was further used in the fed-batch mixed cultivation performed in a 7-L bioreactor. A maximum biomass concentration of 58.6 g L-1 and maximum lipid content of 31.2% w/w DCW were obtained in the fed-batch cultivation. PBWW supplemented with SCM was successfully used as a low-cost medium to produce lipids and carotenoids in a R. toruloides and T. obliquus mixed culture, with higher productivities than in pure cultures, which can significantly reduce the cost of the biofuels obtained.

Impact of High-Pressure Homogenization on the Cell Integrity of Tetradesmus obliquus and Seed Germination.

Microalgae have almost unlimited applications due to their versatility and robustness to grow in different environmental conditions, their biodiversity and variety of valuable bioactive compounds. Wastewater can be used as a low-cost and readily available medium for microalgae, while the latter removes the pollutants to produce clean water. Nevertheless, since the most valuable metabolites are mainly located inside the microalga cell, their release implies rupturing the cell wall. In this study, Tetradesmus obliquus grown in 5% piggery effluent was disrupted using high-pressure homogenization (HPH). Effects of HPH pressure (100, 300, and 600 bar) and cycles (1, 2 and 3) were tested on the membrane integrity and evaluated using flow cytometry and microscopy. In addition, wheat seed germination trials were carried out using the biomass at different conditions. Increased HPH pressure or number of cycles led to more cell disruption (75% at 600 bar and 3 cycles). However, the highest increase in wheat germination and growth (40-45%) was observed at the lowest pressure (100 bar), where only 46% of the microalga cells were permeabilised, but not disrupted. Non-treated T. obliquus cultures also revealed an enhancing effect on root and shoot length (up to 40%). The filtrate of the initial culture also promoted shoot development compared to water (21%), reinforcing the full use of all the process fractions. Thus, piggery wastewater can be used to produce microalgae biomass, and mild HPH conditions can promote cell permeabilization to release sufficient amounts of bioactive compounds with the ability to enhance plant germination and growth, converting an economic and environmental concern into environmentally sustainable applications.

Rhodosporidium toruloides and Tetradesmus obliquus Populations Dynamics in Symbiotic Cultures, Developed in Brewery Wastewater, for Lipid Production.

In this work, primary brewery wastewater (PBWW) and secondary brewery wastewater (SBWW) separately, or mixed at the ratios of 1:1 (PBWW:SBWW) and 1:7 (PBWW:SBWW), with or without supplementation with sugarcane molasses (SCM), were used as culture media for lipid production by a mixed culture of the oleaginous yeast Rhodosporidium toruloides NCYC 921 and the microalgae Tetradesmus obliquus (ACOI 204/07). Flow cytometry was used to understand the dynamics of the two micro-organisms during the mixed cultures evolution, as well as to evaluate the physiological states of each micro-organism, in order to assess the impact of the different brewery effluent media composition on the microbial consortium performance. Both brewery wastewaters (primary and secondary) without supplementation did not allow R. toruloides heterotrophic growth. Nevertheless, all brewery wastewater media, with and without SCM supplementation, allowed the microalgae growth, although the yeast was the dominant population. The maximum total biomass concentration of 2.17 g L-1 was achieved in the PBWW mixed cultivation with 10 g L-1 of SCM. The maximum lipid content (14.86% (w/w DCW)) was obtained for the mixed culture developed on SBWW supplemented with 10 g L-1 of SCM. This work demonstrated the potential of using brewery wastewater supplemented with SCM as a low-cost culture medium to grow R. toruloides and T. obliquus in a mixed culture for brewery wastewater treatment with concomitant lipid production.

CO2 utilization in the production of biomass and biocompounds by three different microalgae.

The atmospheric CO2 increase is considered the main cause of global warming. Microalgae are photosynthetic microorganisms that can help in CO2 mitigation and at the same time produce value-added compounds. In this study, Scenedesmus obliquus, Chlorella vulgaris, and Chlorella protothecoides were cultivated under 0.035 (air), 5 and 10% (v/v) of CO2 concentrations in air to evaluate the performance of the microalgae in terms of kinetic growth parameters, theoretical CO2 biofixation rate, and biomass composition. Among the microalgae studied, S. obliquus presented the highest values of specific growth rate (µ = 1.28 d-1), maximum productivities (P max = 0.28 g L-1d-1), and theoretical CO2 biofixation rates (0.56 g L-1d-1) at 10% CO2. The highest oil content was found at 5% CO2, and the fatty acid profile was not influenced by the concentration of CO2 in the inflow gas mixture and was in compliance with EN 14214, being suitable for biodiesel purposes. The impact of the CO2 on S. obliquus cells' viability/cell membrane integrity evaluated by the in-line flow cytometry is quite innovative and fast, and revealed that 86.4% of the cells were damaged/permeabilized in cultures without the addition of CO2.

Effect of Medium pH on Rhodosporidium toruloides NCYC 921 Carotenoid and Lipid Production Evaluated by Flow Cytometry.

The effect of the culture medium pH (3.5-6.0) on the carotenoid and lipid (as fatty acids) production by the yeast Rhodosporidium toruloides NCYC 921 was studied. Flow cytometry was used to evaluate the yeast's physiological response to different culture medium pH values. The yeast biomass concentration and lipid content were maxima at pH 4.0 (5.90 g/L and 21.85 % w/w, respectively), while the maximum carotenoid content (63.37 µg/g) was obtained at pH 5.0. At the exponential phase, the yeast cell size and internal complexity were similar, at different medium pH. At the stationary phase, the yeast cell size and internal complexity decreased as the medium pH increased. At the exponential phase, the proportion of cells with polarized membranes was always high (>80 %) but at the stationary phase, the proportion of yeast cells with depolarized membranes was dominant (>65 %) and increased with the medium pH increase. The results here reported may contribute for yeast bioprocesses optimization. For the first time, multiparameter flow cytometry was used to evaluate the impact of medium pH changes on the yeast cell physiological status, specifically on the yeast membrane potential, membrane integrity, cell size and internal complexity.

Influence of the carbon source on Gordonia alkanivorans strain 1B resistance to 2-hydroxybiphenyl toxicity.

The viability of bacteria plays a critical role in the enhancement of fossil fuels biodesulfurization efficiency since cells are exposed to toxic compounds such as 2-hydroxybiphenyl (2-HBP), the end product of dibenzothiophene (DBT) biodesulfurization. The goal of this work was to study the influence of the carbon source on the resistance of Gordonia alkanivorans strain 1B to 2-HBP. The physiological response of this bacterium, pre-grown in glucose or fructose, to 2-HBP was evaluated using two approaches: a growth inhibition toxicity test and flow cytometry. The results obtained from the growth inhibition bioassays showed that the carbon source has an influence on the sensitivity of strain 1B growing cells to 2-HBP. The highest IC50 value was obtained for the assay using fructose as carbon source in both inoculum growth and test medium (IC50-48 h = 0.464 mM). Relatively to the evaluation of 2-HBP effect on the physiological state of resting cells by flow cytometry, the results showed that concentrations of 2-HBP >1 mM generated significant loss of cell viability. The higher the 2-HBP concentration, the higher the toxicity effect on cells and the faster the loss of cell viability. In overall, the flow cytometry results highlighted that strain 1B resting cells grown in glucose-SO4 or glucose-DBT are physiologically less resistant to 2-HBP than resting cells grown in fructose-SO4 or fructose-DBT, respectively.

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.

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.

Use of multi-parameter flow cytometry as tool to monitor the impact of formic acid on Saccharomyces carlsbergensis batch ethanol fermentations.

The use of lignocellulosic materials as substrate for bioethanol production is considered a cost-effective approach to make the biofuel production process economically sustainable. However, lignocellulosic hydrolysis releases toxic compounds such as weak acids which inhibit microorganism growth and ethanol production. In order to understand the physiological response of Saccharomyces carlsbergensis when fermenting glucose in the presence of formic acid (HF), the yeast growth was monitored by multi-parameter flow cytometry. Cytoplasmic membrane potential decreased as the HF concentration increased and as the yeast culture reached the stationary phase. However, the proportion of cells with permeabilized membrane did not increase with the HF concentration increase. The accumulation of reactive oxygen species was also monitored. Control and fermentations at low HF concentrations (<1 g/L) resulted in a high proportion of highly oxidized cells at the stationary phase. The multi-parameter flow cytometry approach proved to be a useful tool to monitor the physiological stress response of S. carlsbergensis growth and ethanol production in the presence of HF, an inhibitor present in lignocellulosic hydrolysates. The information here obtained at near real time can be used to enhance second-generation bioethanol production process efficiency.

Effect of acetic acid on Saccharomyces carlsbergensis ATCC 6269 batch ethanol production monitored by flow cytometry.

Bioethanol produced from lignocellulosic materials has been considered a sustainable alternative fuel. Such type of raw materials have a huge potential, but their hydrolysis into mono-sugars releases toxic compounds such as weak acids, which affect the microorganisms' physiology, inhibiting the growth and ethanol production. Acetic acid (HAc) is the most abundant weak acid in the lignocellulosic materials hydrolysates. In order to understand the physiological changes of Saccharomyces carlsbergensis when fermenting in the presence of different acetic acid (HAc) concentrations, the yeast growth was monitored by multi-parameter flow cytometry at same time that the ethanol production was assessed. The membrane potential stain DiOC(6)(3) fluorescence intensity decreased as the HAc concentration increased, which was attributed to the plasmic membrane potential reduction as a result of the toxic effect of the HAc undissociated form. Nevertheless, the proportion of cells with permeabilized membrane did not increase with the HAc concentration increase. Fermentations ending at lower external pH and higher ethanol concentrations depicted the highest proportions of permeabilized cells and cells with increased reactive oxygen species levels. Flow cytometry allowed monitoring, near real time (at-line), the physiological states of the yeast during the fermentations. The information obtained can be used to optimize culture conditions to improve bioethanol production.

Monitoring Rhodosporidium toruloides NCYC 921 batch fermentations growing under carbon and nitrogen limitation by flow cytometry.

The yeast Rhodosporidium toruloides NCYC 921 was grown on carbon or nitrogen limited batch cultures. The fermentations were monitored using traditional techniques and multi-parameter flow cytometry. The lipid content was assessed by flow cytometry in association with the fluorocrome Nile Red which emits yellow gold fluorescence when dissolved in neutral lipids and red fluorescence when dissolved in polar lipids. In this way, it was possible to at-line monitor the yeast lipid composition in terms of polarity classes throughout the batch growths. It was found that the neutral lipids decreased during the carbon-limited stationary phase, and increased during the nitrogen-limited batch growth. The maximum lipid content was obtained for the nitrogen-limited yeast culture (24% w/w lipids). The yeast cells with permeabilised membranes profile remained almost unchanged during the time course of both fermentations. The scatter light measurements (forward and side scatter signals) provided information on the yeast growth phase. The multi-parameter flow cytometric approach here reported represents a better control system based on measurements made at the single cell level for optimization of the yeast lipid production bioprocess performance.

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.

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.

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.

Neochloris oleabundans UTEX #1185: a suitable renewable lipid source for biofuel production.

Energy crises, global warming, and climatic changes call for technological and commercial advances in manufacturing high-quality transportation fuels from unconventional feedstocks. Microalgae is one of the most promising sources of biofuels due to the high yields attained per unit area and because it does not displace food crops. Neochloris oleabundans (Neo) microalga is an important promising microbial source of single-cell oil (SCO). Different experimental growth and lipid production conditions were evaluated and compared by using optical density (540 nm), dry-weight determination, and flow cytometry (FC). Best Neo average biomass productivity was obtained at 30 degrees C under conditions of nitrogen-sufficiency and CO(2) supplementation (N+/30 degrees C/CO(2)), with an average doubling time of 1.4 days. The second and third highest productivities occurred with N-sufficient cultures without CO(2) supplementation at 26 degrees C (N+/26 degrees C) and at 30 degrees C (N+/30 degrees C), with doubling times of 1.7 and 2.2 days, respectively. Microbial lipid production was monitored by flow cytometry using Nile red (NR), a lipophilic fluorochrome that possesses several advantageous characteristics for in situ screening near real time (at line). Results showed maximum lipid content (56%) after 6 days of nitrogen depletion under nitrogen starvation without CO(2) supplementation (N-/30 degrees C), followed by N-/30 degrees C/CO(2) and N-/26 degrees C conditions with 52% lipid content, after 5 and 6 days of N starvation, respectively. The adequate fatty acid profile and iodine value of Neo lipids reinforced this microalga as a good source of SCO, in particular for use as biodiesel.

Oil production towards biofuel from autotrophic microalgae semicontinuous cultivations monitorized by flow cytometry.

Two microalgae species (Scenedesmus obliquus and Neochloris oleoabundans) were cultivated in closed sleeve photobioreactors in order to select the best oil producer for further large-scale open raceway pond cultivations, aiming at biofuel production. Scenedesmus obliquus reached a higher maximum biomass concentration (1.41 g l(-1)) with a lower lipid content (12.8% w/w), as compared to N. oleoabundans [maximum biomass concentration of 0.92 g l(-1) with 16.5% (w/w) lipid content]. Both microalgae showed adequate fatty acid composition and iodine values as substitutes for diesel fuel. Based on these results, N. oleoabundans was selected for further open raceway pond cultivations. Under these conditions, N. oleoabundans reached a maximum biomass concentration of 2.8 g l(-1) with 11% (w/w) of lipid content. 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 both microalgae, making this method a suitable and quick technique for the screening of microalgae strains for lipid production and optimization of biofuel production bioprocesses. Medium growth optimization for enhancement of microalgal oil production is now in progress.

Effect of n-dodecane on Crypthecodinium cohnii fermentations and DHA production.

The potential use of n-dodecane as an oxygen vector for enhancement of Crypthecodinium cohnii growth and docosahexaenoic acid (DHA) production was studied. The volumetric fraction of oxygen vector influenced the gas-liquid volumetric mass transfer coefficient k (L) a positively. The k (L) a increased almost linearly with the increase of volumetric fraction of n-dodecane up to 1%. The stirring rate showed a higher influence on the k (L) a than the aeration rate. The effects of this hydrocarbon on C. cohnii growth and DHA production were then investigated. A control batch fermentation without n-dodecane addition (CF) and a batch fermentation where n-dodecane 1% (v/v) was added (DF) were carried out simultaneously under the same experimental conditions. It was found that, before 86.7 h of fermentation, the biomass concentration, the specific growth rate, the DHA, and total fatty acids (TFA) production were higher in the CF. After this fermentation time, the biomass concentration, the DHA and TFA production were higher in the DF. The highest DHA content of biomass (6.14%), DHA percentage of TFA (51%), and DHA production volumetric rate r (DHA) (9.75 mg l(-1 )h(-1)) were obtained at the end of the fermentation with n-dodecane (135.2 h). The dissolved oxygen tension (DOT) was always higher in the DF, indicating a better oxygen transfer due to the oxygen vector presence. However, since the other C. cohnii unsaturated fatty acids percentages did not increase with the oxygen availability increase due to the n-dodecane presence, a desaturase oxygen-dependent mechanism involved in the C. cohnii DHA biosynthesis was not considered to explain the DHA production increase. A selective extraction through the n-dodecane was suggested.

The use of multi-parameter flow cytometry to study the impact of limiting substrate, agitation intensity, and dilution rate on cell aggregation during Bacillus licheniformis CCMI 1034 aerobic continuous culture fermentations.

The main objective of this work was to establish those factors either physical (power input) or chemical (limiting substrate or dilution rate) that enhance cell aggregation (biofilm or floc formation) and cell physiological state during aerobic continuous cultures of Bacillus licheniformis. Glucose-limited steady-state continuous cultures growing at a dilution rate between 0.64 and 0.87/h and 1,000 rpm (mean specific energy dissipation rate (epsilonT) = 6.5 W/kg), led to the formation of a thin biofilm on the vessel wall characterized by the presence of a high proportion of healthy cells in the broth (after aggregate disruption by sonication) defined as having intact polarized cytoplasmic membranes. An increased epsilonT (from 6.5 W/kg to 38 W/kg) was found to hinder cell aggregation under carbon limitation. The carbon recovery calculated from glucose indicated that additional extracellular polymer was being produced at dilution rates >0.87/h. B. licheniformis growth under nitrogen limitation led to floc formation which increased in size with dilution rate. Counter-intuitively the flocs became more substantial with an increase in epsilonT from 6.5 W/kg to 38 W/kg under nitrogen limitation. Indeed the best culture conditions for enhanced metabolically active cell aggregate formation was under nitrogen limitation at epsilonT = 6.5 W/kg (leading to floc formation), and under carbon limitation at a dilution rate of between 0.64 and 0.87/h, at epsilonT = 6.5 W/kg (leading to vessel wall biofilm formation). This information could be used to optimize culture conditions for improved cell aggregation and hence biomass separation, during thermophilic aerobic bioremediation processes.

Monitoring population dynamics of the thermophilic Bacillus licheniformis CCMI 1034 in batch and continuous cultures using multi-parameter flow cytometry.

Multi-parameter flow cytometry was used to monitor the population dynamics of Bacillus licheniformis continuous cultivations and the physiological responses to a starvation period and a glucose pulse. Using a mixture of two specific fluorescent stains, DiOC6(3) (3,3'-dihexylocarbocyanine iodide), and PI (propidium iodide), flow cytometric analysis revealed cell physiological heterogeneity. Four sub-populations of cells could be easily identified based on their differential fluorescent staining, these correspond to healthy cells (A) stained with DiOC6(3); cells or spores with a depolarised cytoplasmic membrane (B), no staining; cells with a permeabilised depolarised cytoplasmic membrane (C), stained with PI; and permeablised cells with a disrupted cytoplasmic membrane 'ghost cells' (D), stained with both DiOC6(3) and PI. Transmission electron micrographs of cells starved of energy showed different cell lysis process stages, highlighting 'ghost cells' which were associated with the double stained sub-population. It was shown, at the individual cell level, that there was a progressive inherent fluctuation in physiological heterogeneity in response to changing environmental conditions. All four sub-populations were shown to be present during glucose-limited continuous cultures, revealing a higher physiological stress level when compared with a glucose pulsed batch. A starvation period (batch without additional nutrients) increased the number of cells in certain sub-populations (cells with depolarised cytoplasmic membranes and cells with permeabilised depolarised cytoplasmic membranes), indicating that such stress may be caused by glucose limitation. Such information could be used to enhance process efficiency.