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

Jerusalem artichoke as low-cost fructose-rich feedstock for fossil fuels desulphurization by a fructophilic bacterium

Aims: Through biodesulphurization (BDS) is possible to remove the sulphur present in fossil fuels to carry out the very strict legislation. However, this biological process is limited by the cost of the culture medium, and thus, it is important to explore cheaper alternative carbon sources, such as Jerusalem artichoke (JA). These carbon sources usually contain sulphates which interfere with the BDS process. The goal of this work was to remove the sulphates from Jerusalem artichoke juice (JAJ) through BaCl2 precipitation viewing the optimization of dibenzothiophene (DBT) desulphurization by Gordonia alkanivorans strain 1B. Methods and Results: Using a statistical design (Doehlert distribution), the effect of BaCl2 concentration (0·125–0·625%) and pH (5–9) was studied on sulphate concentration in hydrolysed JAJ. A validated surface response derived from data indicated that zero sulphates can be achieved with 0·5–0·55% (w/v) BaCl2 at pH 7; however, parallel BDS assays showed that the highest desulphurization was obtained with the juice treated with 0·5% (w/v) BaCl2 at pH 8·73. Further assays demonstrated that enhanced DBT desulphurization was achieved using hydrolysed JAJ treated in these optimal conditions. A total conversion of 400 µmol l-1 DBT into 2-hydroxybiphenyl (2-HBP) in <90 h was observed, attaining a 2-HBP maximum production rate of 28·2 µmol l-1 h-1 and a specific production rate of 5·06 µmol-1 g-1(DCW) h-1. Conclusions: These results highlight the efficacy of the treatment applied to JAJ in making this agromaterial a promising low-cost renewable feedstock for improved BDS by the fructophilic strain 1B. Significance and Impact of the Study: This study is a fundamental step viewing BDS application at the industrial level as it accounts a cost-effective production of the biocatalysts, one of the main drawbacks for BDS scale-up

Influence of culture conditions towards optimal carotenoid production by Gordonia alkanivorans strain 1B

ABSTRACT: With the increasing awareness on the toxicity of several synthetic dyes, demand for pigments from natural sources, such as microbial carotenoids, has gained interest as a promising safe alternative colour additive. In this study, a surface response methodology based on the Doehlert distribution for two factors [% of glucose in a mixture of glucose + fructose (10 g/L total sugars), and sulfate concentration] was used towards the optimal carotenoids production by Gordonia alkanivorans strain 1B in the presence of light (400 lx). Time influence on pigment production by this bacterium was also evaluated, as well as the cell viability profile during longer incubation periods at optimal conditions. Indeed, the highest carotenoid production (2596-3100 mu g/g(DCW)) was obtained when strain 1B was cultivated in the optimal conditions: glucose 10 g/L and sulfate >= 22 mg/L, in the presence of light for 19 days at 30 degrees C, 150 rpm. Flow cytometry showed that the highest production was somehow related with the cellular stress. These results highlight the great potential of strain 1B as a new hyperpigment producer to be exploited towards several applications.info:eu-repo/semantics/publishedVersio

Isolation and identification of Magnusiomyces capitatus as a lipase-producing yeast from olive mill wastewater

ABSTRACT: Olive mill wastewaters (OMW) are effluents originated from olive oil extraction. As an oil-rich residue, OMW is a potential source of lipase-producing microorganisms and a complex medium potentially suitable for lipase production. The aim of the present study was to isolate yeasts with the ability to produce extracellular lipases from OMW. Thirty-two yeast isolates were obtained and screening for esterase/lipase activity using rapid plate detection methods allowed the selection of five isolates. Subsequently, extracellular lipolytic activity was determined in shake-flasks, and the best activity was found in the isolate JT5 (0.85 U/mL). This isolate was identified as Magnusiomyces capitatus by DNA sequencing. Growth and lypolytic activities by M. capitatus JT5 were assessed in undiluted OMW, and optimization of lipase production was achieved by a positive interaction of two factors (oxygen availability and nitrogen concentration). The highest lipase activity (1.4 U/mL) was obtained at NH4Cl concentration of 2.8 g/L and kLa of 0.65 min−1. The growth of M. capitatus JT5 in a stirred tank bioreactor, using undiluted OMW, allowed the improvement of lipase production (up to 3.96 U/mL) by increasing olive oil concentration in the medium, under the selected conditions of nitrogen concentration and oxygen availability. This study highlighted the isolate M. capitatus JT5 as a lipase-producing microorganism that is able to grow in undiluted OMW under controlled conditions. Results obtained in shake-flasks have been reproduced satisfactorily in the stirred tank bioreactor.info:eu-repo/semantics/publishedVersio

Surface response methodology towards optimal carotenoids production by Gordonia Alkanivorans Strain 1B [Poster]

ABSTRACT: The process of obtaining carotenoids, mainly towards sectors that may influence the human health, such as pharmaceutical and cosmetic, is strictly regulated because of the potential toxicity of the synthetically derived pigments. Thus, microbial pigments are in increasing demand since they are a promising natural and safe alternative source for various industrial applications. Gordonia alkanivorans strain 1B is a fructophilic desulfurizing bacterium, which was also shown to be a good producer of carotenoids. However, its production abilities presented a great variation, depending on the conditions it was submitted to. In previous works, both the carbon source and sulfur source, demonstrated a great influence in the total carotenoid concentration, especially when combined with the presence of a light source. So, in this study, a surface response methodology based on the Doehlert distribution for two factors (% of glucose in a mix glucose + fructose (10 g/L total sugars), and sulfate concentration) was used aiming to get the optimal carotenoids production by G. alkanivorans strain 1B.N/

Carbon concentration and oxygen availability affect lipid and carotenoid production by carob pulp syrup-grown Rhodosporidium toruloides NCYC 921

The simultaneous effect of oxygen availability and carbon source concentration on yeast lipid and carotenoid production has never been studied before. In this work, a Doehlert distribution design was used to study the simultaneous effect of carbon concentration and oxygen availability on Rhodosporidium toruloides NCYC 921 carotenoid and lipid production. A cheap industrial byproduct was used as carbon source (carob pulp syrup). A total sugar concentration of 106.3 g/L and a medium volume of 0.120 L induced the highest total carotenoid and total fatty acid productivities (4.60 ìg/Lh and 0.029 g/Lh, respectively). Flow cytometry was used to assess yeast stress response under different cultivation conditions. The highest proportion of cells with permeabilised membrane (>20%) was induced when the cultivations were carried out at the highest sugar concentration studied (130.0 g/L) or when the culture reached the minimum final medium pH (4.60). The results showed that the total sugar concentration had a positive influence on the yeast biomass and carotenoid content, while the oxygen availability had little influence on the biomass concentration, but had a slight positive influence on the carotenoid content. Regarding the fatty acids, the two factors had a negative impact on the synthesis of these compounds

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 L1 induced the highest total fatty acid productivity (1.90 g L1 h1) and the highest carotenoid productivity (9.79 lg L1 h1). 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

Ionic liquids toward enhanced carotenoid extraction from bacterial biomass

ABSTRACT: Carotenoids are high added-value products primarily known for their intense coloration and high antioxidant activity. They can be extracted from a variety of natural sources, such as plants, animals, microalgae, yeasts, and bacteria. Gordonia alkanivorans strain 1B is a bacterium recognized as a hyper-pigment producer. However, due to its adaptations to its natural habitat, hydrocarbon-contaminated soils, strain 1B is resistant to different organic solvents, making carotenoid extraction through conventional methods more laborious and inefficient. Ionic liquids (ILs) have been abundantly shown to increase carotenoid extraction in plants, microalgae, and yeast; however, there is limited information regarding bacterial carotenoid extraction, especially for the Gordonia genus. Therefore, the main goal of this study was to evaluate the potential of ILs to mediate bacterial carotenoid extraction and develop a method to achieve higher yields with fewer pre-processing steps. In this context, an initial screening was performed with biomass of strain 1B and nineteen different ILs in various conditions, revealing that tributyl(ethyl)phosphonium diethyl phosphate (IL#18), combined with ethyl acetate (EAc) as a co-solvent, presented the highest level of carotenoid extraction. Afterward, to better understand the process and optimize the extraction results, two experimental designs were performed, varying the amounts of IL#18 and EAc used. These allowed the establishment of 50 µL of IL#18 with 1125 µL of EAc, for 400 µL of biomass (cell suspension with about 36 g/L), as the ideal conditions to achieve maximal carotenoid extraction. Compared to the conventional extraction method using DMSO, this novel procedure eliminates the need for biomass drying, reduces extraction temperatures from 50 °C to 22 ± 2 °C, and increases carotenoid extraction by 264%, allowing a near-complete recovery of carotenoids contained in the biomass. These results highlight the great potential of ILs for bacterial carotenoid extraction, increasing the process efficiency, while potentially reducing energy consumption, related costs, and emissions.info:eu-repo/semantics/publishedVersio

Physiological effects of the addition of n-dodecane as an oxygen vector during steady-state Bacillus licheniformis thermophillic fermentations perturbed by a starvation period or a glucose pulse

The effect of the presence of n-dodecane as a potential oxygen vector during oxygen limited continuous cultures of a Bacillus strain was studied, under extreme nutrient supply conditions: glucose excess, limitation and starvation. The addition of n-dodecane to the aqueous phase of a mechanically agitated and aerated fermentation increased the kLa by up to 35%. The n-dodecane additions to B. licheniformis cells during starvation (oxygen limitation with concomitant glucose starvation) caused a severe detrimental progressive change in cell physiological state with respect to cytoplasmic membrane polarisation and permeability which was mitigated against by alleviating either the oxygen limitation (by increasing the mean energy dissipation rate or by the addition of n-dodecane as an oxygen vector) or by alleviating the carbon limitation (by resuming the carbon feed or by the addition of a glucose pulse). Further that during periods of excess glucose (glucose pulse) a much higher kLa was required to prevent the onset of anaerobic mixed acid fermentation than could be provided by the addition of n-dodecane alone. N-dodecane can be used to increase the kLa when added in sufficient quantities to the aqueous phase of a mechanically agitated and aerated bioreactor but the magnitude of this increase is process and vessel geometry specific