Search

Lignocellulosic materials have been considered low-cost effective substrates for bioethanol production. However, lignocellulosic pretreatment releases toxic compounds such as 5-hydroxymethylfurfural (HMF) that is known to inhibit the yeast growth and ethanol production. In this work, flow cytometry was used to monitor the physiological response of the yeast Saccharomyces carlsbergensis ATCC 6269 in the presence of different initial HMF concentrations within the range of 0-15 g/L, in terms of cell membrane integrity, potential, and intracellular lipids. It was observed that the HMF presence affected more significantly the yeast growth than the ethanol production. At 15 g/L HMF, the yeast growth and fermentation ability were completely inhibited. The cell membrane integrity and potential decreased as the initial HMF concentration increased. At the end of the fermentation process with 10 g/L HMF, the yeast culture contained 45 % of cells with depolarized plasma membrane, 52 % of cells with permeabilized plasma membrane, and 53 % of cells with increasing reactive oxygen species (ROS) levels. Using the Nile Red stain, it was observed that intracellular polar lipids were more affected by the initial HMF concentration than the neutral lipids, probably due to the extensive membrane damage.

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.

Flow cytometry was used to assess β-carotene content, cell membrane permeability, cell size and granularity in Rhodotorula glutinis mutant 400A15 grown under different oxygen transfer coefficients (k L a) and carbon to nitrogen ratios (C/N). A Doehlert distribution was used in order to select the best conditions that induced the highest carotenoids production. The highest β-carotene content (0.79 mg g(-1) DCW) at the lowest k L a and C/N (5 × 10(-3) s(-1) and 11.3 respectively). Under these conditions, the biomass concentration attained 18.60 g L(-1). The highest ratio of cells with permeabilised membranes (2.6 %), and the highest cell size and granularity were also obtained under these conditions. It was observed that C/N showed a stronger influence than the k L a on the measured cell parameters.

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.

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., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

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.

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.

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.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 ( Pmax = 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. [ABSTRACT FROM AUTHOR]

Abstract: Carbon limited continuous cultures of Lactobacillus rhamnosus ATCC 7469 were grown at dilution rates between 0.1h−1 and 0.6h−1. At 0.45h−1, oxygen uptake decreases producing a deficiency in the production of cell energy, lowering the concentration of biomass and finally accumulating glucose in the broth. Under the lack of energy pressure, L. rhamnosus ATCC 7469 triggers the production of lactic acid from pyruvate freeing NAD+ and stimulates glycolysis to continue, producing extra ATP from substrate-level phosphorylation. The 12-fold growing concentration of lactic acid and the 2-fold increase of succinic acid are in parallel with the steep 4-fold decrease of acetic acid production and small concentration changes of formic and propionic acids. The way the cells balance the available energy between the growing dilution rate and detoxification produces a stress within the culture, detected and described by flow cytometry. As the dilution rate increased, the proportion of L. rhamnosus ATCC 7469 cells with depolarized membrane steadily increased (1% at D =0.20h−1, 8% at D =0.30h−1, 14% at D =0.45h−1 and 26% for D =0.62h−1, respectively). Only a low level of 3.7% of the population did not recover from the demanding growth rates in the acidic environment. [Copyright &y& Elsevier]

Abstract: This work demonstrates, for the first time, the potential of using multi-parameter flow cytometry to monitor changes in the microbial cytoplasmic membrane integrity and polarization during microbial fuel cells (MFC) operation. Such information is crucial to follow the dynamics of bacteria colonization of the electrodes and their viability maintenance during electrical current production. Interestingly, the results show that during voltage production, the electrostatic gradients of the bacteria cytoplasmic membrane are disturbed, leading to depolarization of a subpopulation (where less than 40% of the cells were polarized). Once the voltage dropped, due to substrate limitation, several cells in the anode supernatant restored their polarized state. This process was reversible and observed over more than 4 cycles of fresh substrate addition. Similar power outputs induced similar membrane polarization results, regardless of the substrate used. The percentage of non-viable cells was maintained constant during current production. This study opens new opportunities to monitor cell behavior, and thus increase the knowledge of dynamic mechanisms responsible for current production at the individual cell level. This technique could be of great interest for the development of new MFC configurations and optimization of MFC operation conditions toward increased performance. [Copyright &y& Elsevier]

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. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

Objectives. CD38 participates in lymphocyte ontogeny and function and may be involved in autoimmunity. Absence of CD38 accelerates development of non-obese diabetic (NOD) mice diabetes and anti-CD38 antibodies are good markers of human disease. Little is known regarding systemic autoimmunity. Active SLE patients have higher numbers of CD38+ T and B cells. CD38 is a candidate gene for the murine Lmb2 lupus locus. We aimed to investigate whether CD38 was involved in lupus development.Methods. We developed Cd38−/−-Faslpr/Faslpr mice and monitored them for development of a lupus-like disease through measurement of protein excretion in urine, histological assessment of the kidneys, quantification of circulating immunoglobulins and autoantibodies. We have also immunophenotyped 2- and 6-month old Cd38−/−-Faslpr/Faslpr mice.Results. We found that absence of CD38 accelerated disease development: female Cd38−/−-Faslpr/Faslpr mice presented severe proteinuria, GN, deposition of ICs in the renal medulla and increased amounts of circulating immunoglobulin G (IgG), although anti-dsDNA autoantibodies and RF were not significantly increased at disease onset. We have found that Cd38−/−-Faslpr/Faslpr male mice, similarly to other murine models of lupus, were able to control disease. Absence of CD38 in lpr mice altered differentiation of T cells and dendritic cells (DC).Conclusion. Although the role of CD38 in tolerance is still to be elucidated, we provide evidence that it may play an active role in the control of a murine lupus-like disease. [ABSTRACT FROM PUBLISHER]

Abstract: 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: During microbial bioprocesses development, it is crucial monitoring cell stress response as such information determines the process efficiency. Multi-parameter flow cytometry coupled with fluorescent stains [PI and DiOC6(3) (3,3′-dihexylocarbocyanine iodide)] was used to monitor bacterial batch cultures. It was observed that the most significant differences in the cell physiological responses of these microorganisms were observed at the early stationary phase. While Bacillus licheniformis batch culture displayed 91% of healthy cells, 1.3% of cells with depolarised cytoplasmic membrane and 8% of cells with both depolarised and permeabilised cytoplasmic membrane as the culture reached this phase, Lactobacillus rhamnosus batch culture displayed 66% of healthy cells, 29% of cells with depolarised cytoplasmic membrane and 5% of depolarised and permeabilised cytoplasmic membrane, at that stage. However, at the late stationary phase, both bacterial cultures showed a high proportion of polarised healthy cells, suggesting that the microorganisms could be using storage materials as a survival strategy. It was found that the double staining protocol using PI and DiOC6(3) could be used to monitor cell physiological response of both bacterial batch cultures and provided important physiological information at the individual cell level that is difficult to obtain in any other way, which can be used to enhance bioprocesses efficiency. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

Abstract: Multi-parameter flow cytometry (MPFC) coupled with specific fluorescent stains were used to monitor the physiological response of individual cells to starvation periods as well as lactose and glucose pulses during the steady state of a continuous culture of Bacillus licheniformis. Measurements of off-gas (CO2 produced and O2 consumed) and optical density measurements were also made and used to follow bulk biomass proliferation and metabolic activity of the microbial population. It was clear that the physiological response to each perturbation was different in type and extent dependent on the conditions experienced and that these responses could be followed by the techniques employed. The use of MPFC, a rapid, real time analysis of individual microbial cells, is enhancing our knowledge of how different physiological sub-populations develop with time (population dynamics) throughout a bio-process further demonstrating the inherent complex heterogeneity of microbial cultures even during the so called ‘steady-state’ of a continuous culture fermentation. These results are discussed in terms of devising better control systems based on measurements made at the individual cell level for optimisation of bioprocess performance. [Copyright &y& Elsevier]

Crude glycerol, a biodiesel industry byproduct, and corn steep liquor (CSL) derived from a starch industry, were used as carbon and nitrogen sources, respectively, for lipid production, using the heterotrophic microalga C. cohnii grown in a bench bioreactor, in a batch culture. The maximum biomass concentration, lipid content and lipid productivity attained were 5.34 g/L, 24.6% (w/w Dry Cell Weight-DCW) and 0.016 g L−1 h−1, respectively. Flow cytometry analysis was used to evaluate the impact of these substrates on the microalgae cells. A high proportion of intact cells with enzymatic (esterases) activity (>50%) was present throughout the cultivation time course. These results indicate that crude glycerol and CSL can be used in the medium formulation for DHA and lipid production using this microalga, which reduce the process costs in an expected maximum of 84%. [ABSTRACT FROM AUTHOR]

Brewery effluents supplemented with sugarcane molasses and urea were used as growth medium to develop the yeast Rhodosporidium toruloires NCYC 921, to produce triacylglicerols (TAGs) and carotenoids. The yeast cultivation was monitored by flow cytometry, in order to understand the impact of the effluent on the yeast cell membrane. Ideally the treated effluent may be discharged in the water bodies. • Brewery effluent was used to grow R. toruloides for lipids and carotenoids production. • Sugarcane molasses and urea improved the yeast lipids and carotenoids production. • Flow cytometry was successfully used to monitor the yeast cell membrane integrity. In this study, secondary brewery wastewater (SBWW) supplemented with sugarcane molasses (SCM) was used for SBWW treatment with concomitant lipid and carotenoid production by the yeast Rhodosporidium toruloides NCYC 921. In order to improve the biomass production, ammonium sulfate, yeast extract and urea were tested as nitrogen sources. Urea was chosen as the best low-cost nitrogen source. A fed-batch cultivation was carried out with SBWW supplemented with 10 g L−1 of sugarcane molasses as carbon source, and 2 g L−1 of urea as nitrogen source. A maximum biomass concentration of 42.5 g L−1 was obtained at t = 126.5 h and the maximum biomass productivity was 0.55 g L−1 h−1 at t = 48.25 h. The maximum lipid content was 29.9 % w/w (DCW) at t = 94 h of cultivation and the maximum carotenoid content was 0.23 mg g−1 at 120 h of cultivation. Relatively to the SBWW treatment, after the batch phase, 45.8 % of total Kjeldahl nitrogen removal, 81.7 % of COD removal and 100 % of sugar consumption were observed. Flow cytometry analysis revealed that 27.27 % of the cells had injured membrane after the inoculation. This proportion was reduced to 10.37 % at the end of the cultivation, indicating that cells adapted to the growth conditions. [ABSTRACT FROM AUTHOR]

This study evaluated the operation of a hybrid anaerobic reactor fed with algal biomass cultivated in effluent from the brewery industry. Three stages of operation were distinguished during the 72 days of semi-continuous functioning of the reactor: Stage 1 (S1), in which algal biomass was used as substrate; Stage 2 (S2), in which 10% (v/v) of the algal biomass was substituted by olive mill wastewater (OMW); and Stage 3 (S3), in which algal biomass was heat pre-treated. During S1, a loss of solids was observed, with an increment of organic matter in the outlet. The substitution of 10% of the volume of algal biomass by OMW tripled the methane productivity obtained in the previous stage by digestion of pure algal biomass. Heat pre-treatment was not efficient in rupturing the cell wall, and consequently did not have any effect on the increase in biogas production. The complementarity of substrates in the assessed conditions led to better results than the pre-treatment of the algal biomass. [ABSTRACT FROM AUTHOR]

Yeast production and biomass biorefinery processes for lipid and carotenoid extraction generate residues that can be used as substrates for anaerobic digestion. Glucose and carob pulp syrups were used as carbon sources to produce the yeast biomass. The yeast cultivation broth, yeast biomass residues (after carotenoid and lipid extraction) and the carob pulp solid residues obtained from the extraction of sugars were used to produce biogas by applying different Substrate/Inoculum ratios (S/I of 0.5 and 0.75). For all the residues studied, the digestions at the S/I ratio of 0.75 provided higher biogas yields than those carried out at the S/I ratio of 0.5. The best results in terms of biogas production and methane yield were observed for the yeast residue digestion at S/I of 0.75 (65.9 mL, 333.7 mL g −1 VS −1 substrate). As monitored through flow cytometry, its bacterial consortium showed the lowest proportion of injured cells. [ABSTRACT FROM AUTHOR]

Brewery wastewater (BWW) is an appealing low-cost substrate for the production of single cell oils by oleaginous microorganisms. However, it may contain inhibitor compounds that may affect the microbial metabolism. This work investigated, for the first time, the presence of potential inhibitor compounds in primary brewery wastewater (PBWW) and secondary brewery wastewater (SBWW) for the pure and mixed cultivation of the yeast Rhodosporidium toruloides NCYC 921 and the microalga Tetradesmus obliquus ACOI 204/07. Three organic acids (OrgAc) were identified in the brewery effluents (acetic, propionic and butyric acids). Yeast and microalga pure and mixed cultivations were performed in PBWW and SBWW in order to understand the behaviour of the microorganisms, individually and together. Flow cytometry (FC) was used to monitor each microbial population during the mixed cultivations, and to study the yeast and microalga cell viability throughout all cultivations. The yeast cells in pure cultures grown in both effluents were severely affected by the OrgAc presence confirmed by the cell stress results obtained by FC. However, in the mixed cultures, the yeast cells were able to develop, and the levels of stress conditions were considerably lower. Only in microalga pure and mixed cultures efficient OrgAc removal was observed. • Brewery wastewater (BWW) contains inhibitors that may affect the microbial metabolism. • It was possible to identify the presence of three organic acids (OrgAc) in BWW. • R. toruloides and T. obliquus pure and mixed cultivations were performed using BWW. • R. toruloides pure cultures were severely affected by the OrgAc present in the BWW. • T. obliquus pure and mixed cultures were efficient in removing the OrgAc in the BWW. [ABSTRACT FROM AUTHOR]

Highlights: [•] Carop pulp syrup (CPS) and sugarcane molasses (SCM) were used as carbon sources. [•] CPS at 75gL−1 induced the highest fatty acid and carotenoid productivities. [•] Flow cytometry detected differences between the cell membrane grown on CPS and SCM. [•] R. toruloides growth on CPS induced lower ratio of permeabilised cells than on SCM. [ABSTRACT FROM AUTHOR]

Highlights: [•] Flow cytometry was used to evaluate R. toruloides carotenoids and cell viability. [•] R. toruloides mitochondrial membrane potential was affected by starvation. [•] R. toruloides cell membrane integrity was reduced by starvation. [•] R. toruloides intracellular esterase activity was not affected by starvation. [•] R. toruloides biomass was a promised source of carotenoids and fatty acids. [Copyright &y& Elsevier]