Transfusion of donor-derived red blood cells (RBCs) is the most common form of cell therapy. Production of transfusion-ready cultured RBCs (cRBCs) is a promising replacement for the current, fully donor-dependent therapy. A single transfusion unit, however, contains 2 × 10¹² RBC, which requires large scale production. Here, we report on the scale-up of cRBC production from static cultures of erythroblasts to 3 L stirred tank bioreactors, and identify the effect of operating conditions on the efficiency of the process. Oxygen requirement of proliferating erythroblasts (0.55–2.01 pg/cell/h) required sparging of air to maintain the dissolved oxygen concentration at the tested setpoint (2.88 mg O₂/L). Erythroblasts could be cultured at dissolved oxygen concentrations as low as 0.7 O₂ mg/ml without negative impact on proliferation, viability or differentiation dynamics. Stirring speeds of up to 600 rpm supported erythroblast proliferation, while 1800 rpm led to a transient halt in growth and accelerated differentiation followed by a recovery after 5 days of culture. Erythroblasts differentiated in bioreactors, with final enucleation levels and hemoglobin content similar to parallel cultures under static conditions.

A systematic study on the physicochemical properties of two protic ionic liquids (ILs) {2-hydroxyethylammonium acetate ([Mea][Ac]) and 2-hydroxyethylammonium hexanoate ([Mea][Hex])} and their mixtures with water was performed. The density and viscosity were assessed across the entire range of aqueous dilutions between 278 and 393 K. The conductivities, water activities, and surface tension of the binary systems in water were also assessed, and the influence of anions was evaluated. Differential scanning calorimetry (DSC), Fourier transform infrared (FTIR), and 1H and 13C nuclear magnetic resonance (NMR) techniques were used to study the systems at different IL compositions. The excess molar volumes (VE) and thermal expansion coefficients were calculated, with negative values for VE across the entire concentration range. Density data were fitted to a polynomial for density prediction, function of temperature, and concentration, with the average deviation percentage not exceeding 0.63%. The viscosities of the binary systems were studied considering six different models and were better predicted by the model of Herráez et al. at IL concentrations higher than 0.25 mole fraction. The systems containing [Hex]- exhibited higher water activities and lower conductivity and surface tension. All studied systems exhibited a glass transition event, which varied according to the IL composition. The FTIR and NMR analysis confirmed the distinct molecular arrangement of [Mea][Ac] and [Mea][Hex] systems.

Liquid hot water pretreatment is considered to be a promising method for increaing biomass digestibility due to the moderate operational conditions without chemical additionA necessary step towards the scalability of this pretreatment process is performing pilot plant triaUpscaling was evaluated with a scaling factor of 500, by using 50 mL in the laboratory and 25 L in pilot plant batch reactor. Pretreatment times were varied from 30 to 240 min, and temperatures usewere 180–188^◦ C, while applying similar heating profiles at both scales. The initial mass fraction poplar wood chips ranged from 10% to 16%. Liquid hot water pretreatment at laboratory and pilscale led to analogous results. The acetic acid analysis of the liquid and solid fractions obtained aftpretreatment indicated that complete deacetylation of poplar biomass can be achieved.

The use of lignocellulosic (LC) materials, especially residues, as feedstock in biorefinery applications is a promising alternative to the oil refinery production of fuels, power and chemicals, reducing the global warming potential (GWP) related to these activities. The conversion of LC's carbohydrates into useful sugars is entirely dependent on the efficiency of the pretreatment (PT) step. Ionic liquids (ILs) have been explored as tailored solvents for the solubilization of LC's complex structure during PT, which can overcome the existing hurdles related to PT. This work assesses the impact of PT variables and the IL recycling through freezing concentration (FC) in an IL-based biorefinery. The influence of temperature, solid loading and IL dilution was systematically studied and the mass and energy balances, economic and environmental outcomes calculated. Life cycle analysis (LCA) was employed in a cradle-to-gate approach. Results showed that solid loading and IL dilution, rather than PT temperature, have the major influence on the energy requirements to produce 1 kg of ethanol. IL make up is a critical parameter to minimize the environmental impact and operational costs associated with the process. Product selling price and IL recycling were the most impacting variables concerning profitability. Extra investment for improved IL recycling is advantageous up to 99% of recovery. Product diversification can improve the economic feasibility even if it is associated with increased capital expenditure. The results show the importance of the pretreatment design and solvent recycling from an integrated perspective, thus challenging the criteria defined while assessing these steps alone.

Development of affordable and low carbon biobased manufacturing depends critically on strategies that reduce cost and emission profiles. This paper indicates that efforts around the reduction of capital costs by intensification of process equipment need to be carefully weighed against the inherently fast increasing financial and climate costs of driving forces used for the intensification. The fundamental relation between capital expenditures (CAPEX) and operational expenditures (OPEX) of intensified and non-intensified biobased processes and their financial and climatic impacts are emphasized and provisionally explored for a few industrial processes. General learnings flag the importance in particular of OPEX minimisation for sustainable bio-economic development.

Table 2 in printed publication was miss-set, and should read as follows: [Table presented] The authors would like to apologize for any inconvenience caused.

Biobased production has been promoted as an alternative to fossil-based production to mitigate climate change. However, emerging concerns over the sustainability of biobased products have shown that tensions can emerge between different objectives and concerns, like emission reduction targets and food security, and that these are dependent on local contexts. Here we present the Open Sustainability-in-Design (OSiD) framework, the aim of which is to integrate a context-sensitive sustainability analysis in the conceptual design of biobased processes. The framework is illustrated, taking as an example the production of sustainable aviation fuel in southeast Brazil. The OSiD framework is a novel concept that brings the perspectives of stakeholders and considerations of the regional context to an ex ante sustainability analysis of biobased production. This work also illustrates a way to integrate methods from different scientific disciplines supporting the analysis of sustainability and the identification of tensions between different sustainability aspects. Making these tensions explicit early in the development of biobased production can make them more responsive to emerging sustainability concerns. Considering the global pressure to reduce carbon emissions, situating sustainability analyses in their socio-technical contexts as presented here can help to explain and improve the impacts of biobased production in the transition away from fossil resources.

Intracellular metabolites were evaluated during the continuous growth of Trichoderma harzianum P49P11 under carbon-limited conditions. Four different conditions in duplicate were investigated (10 and 20 g/L of glucose, 5.26/5.26 g/L of fructose/glucose and 10 g/L of sucrose in the feed). Differences in the values of some specific concentrations of intracellular metabolites were observed at steady-state for the duplicates. The presence of extracellular polysaccharide was confirmed in the supernatant of all conditions based on FT-IR and proton NMR. Fragments of polysaccharides from the cell wall could be released due to the shear stress and since the cells can consume them under carbon-limited conditions, this could create an unpredictable carbon flow rate into the cells. According to the values of the metabolite concentrations, it was considered that the consumption of those fragments was interfering with the analysis.

BACKGROUND: Recently, a new process concept has been proposed to selectively adsorb wort off-flavours, i.e. aldehydes, from alcohol-free beers with hydrophobic zeolites. RESULTS: In this work, we investigated the uptake of a mixture of wort flavour compounds (2-methylpropanal, 2-methylbutanal, 3-methylbutanal, furfural, and methional), from a model solution onto binderless hydrophobic ZSM-5 zeolite granules in order to quantify mass transfer parameters and identify bottlenecks. Subsequently, the homogenous solid diffusion model was employed to regress the effective diffusion coefficients for each molecule and experimental condition, which ranged between 10⁻¹⁵ and 10⁻¹³ m² s⁻¹, indicating strong intraparticle mass transfer limitation. Furthermore, it was found that the effective diffusion coefficient is inversely correlated to the molecules' hydrophobicity, expressed as the logD value and its isotherm affinity constant. CONCLUSION: These results give valuable insight to design and improve the adsorbent material and an off-flavour removal unit at industrial scale.

Carbon-limited chemostat cultures were performed using different carbon sources (glucose, 10 and 20 g/L; sucrose, 10 g/L; fructose/glucose, 5.26/5.26 g/L; carboxymethyl cellulose, 10 g/L; and carboxymethyl cellulose/glucose, 5/5 g/L) to verify the capability of the wild type strain Trichoderma harzianum to produce extracellular enzymes. All chemostat cultures were carried out at a fixed dilution rate of 0.05 h⁻¹. Experiments using glucose, fructose/glucose and sucrose were performed in duplicate. Glucose condition was found to induce the production of enzymes that can catalyse the hydrolysis of p-nitrophenyl-β-D-glucopyranoside (PNPGase). A concentration of 20 g/L of glucose in the feed provided the highest productivity (1048 ± 16 U/mol h). Extracellular polysaccharides were considered the source of inducers. Based on the obtained results, a new PNPGase production process was developed using mainly glucose. This process raises interesting possibilities of synthesizing the inducer substrate and the induced enzymes in a single step using an easily assimilated carbon source under carbon-limited conditions.

The wild type strain Trichoderma harzianum was able to synthesize enzymes that can catalyse the hydrolysis of p-nitrophenyl-β-D-glucopyranoside (PNPGase) in glucose-limited chemostat cultures. Fructose/glucose and sucrose conditions provided low levels of PNPGase activity. To investigate whether under these conditions other enzymes were produced, a shotgun proteomics analysis of their supernatants was performed. The analysis has indicated that the different carbon sources used influenced the amounts of proteins secreted including 1,3-beta-glucanosyltransferase, alpha-1,2-mannosidase, alpha-galactosidase and glucan 1,3-beta-glucosidase. The analysis has also suggested the presence of beta-glucosidase, which could also be represented by PNPGase activity. Intracellular metabolites were quantified during PNPGase production for the condition using 20 g/L of glucose in the feed and differences were observed, indicating that intracellular glucose could be inhibiting PNPGase production. Significance: This work shows that sugars such as glucose, fructose/glucose and sucrose can be used as substrates for the continuous synthesis of different enzymes under carbon-limited conditions by Trichoderma harzianum. As far as we know, this is the first work about the continuous synthesis of enzymes under carbon-limited conditions suggesting that different easily assimilated carbon sources can be used to generate different enzymatic cocktails. Each enzyme or uncharacterized protein suggested by shotgun proteomics has the potential to become a promising product for biotechnological applications.

Emulsion formation is a major concern when dealing with multiphasic fermentations. Flocculants can be used together with other demulsification techniques to improve oil recovery in multiphasic fermentations. In this paper, the impact of adding flocculants during a multiphasic fermentation with 10 wt% dodecane, to destabilize the broth emulsion, improve creaming formation and enhance oil recovery is studied. Flocculants, CaCl₂ and (NH₄)₂SO₄ were shown to be the most promising flocculants. Flocculant addition, their time of addition, and its impact on multiphasic fermentations has been evaluated by comparing fermentation performance against reference fermentations and three oil recovery methods: gravity settling, gas enhanced oil recovery and centrifugation. When adding 75 mM of (NH₄)₂SO₄ during fermentation, the creaming rate during gravity settling increased 3-fold and the oil recovery by gas enhanced oil recovery was 35%, without altering fermentation performance. Addition of CaCl₂ during fermentation resulted in 88% and 67% oil recovery for early and late addition, which is a 4 and 3-fold increase in comparison with the reference. Yet, CaCl₂ deviated from standard fermentation performance when added immediately after second phase addition. In conclusion, flocculant addition during multiphasic fermentation can be used to destabilize microbial emulsions and potentially improve in situ oil recovery.

The enzymatic conversion of lignocellulosic material to sugars can provide a carbon source for the production of energy (fuels) and a wide range of renewable products. However, the efficiency of this conversion is impaired due to product (sugar) inhibition. Even though several studies investigate how to overcome this challenge, concepts on the process to conduct the hydrolysis are still scarce in literature. Aqueous two-phase systems (ATPS) can be applied to design an extractive reaction due to their capacity to partition solutes to different phases in such a system. This work presents strategies on how to conduct extractive enzymatic hydrolysis in ATPS and how to explore the experimental results in order to design a feasible process. While only a limited number of ATPS was explored, the methods and strategies described could easily be applied to any further ATPS to be explored. We studied two promising ATPS as a subset of a previously high throughput screened large set of ATPS, providing two configurations of processes having the reaction in either the top phase or in the bottom phase. Enzymatic hydrolysis in these ATPS was performed to evaluate the partitioning of the substrate and the influence of solute partitioning on conversion. Because ATPS are able to partition inhibitors (sugar) between the phases, the conversion rate can be maintained. However, phase forming components should be selected to preserve the enzymatic activity. The experimental results presented here contribute to a feasible ATPS-based conceptual process design for the enzymatic conversion of lignocellulosic material.

Cellulase production can be divided into two steps: growth stage; followed by an induction stage. To develop a mathematical model for the optimization of this strategy, two sets of experiments were performed in batch mode for parameter estimation. One set of experiments was performed to evaluate the influence of glycerol regarding cell growth (initial concentrations of 5, 10, 15 and 20 g/L). The other set of experiments considered the induction stage using cellulose as the substrate (initial concentrations of 5, 10, 20, 30 and 40 g/L). Two feeding strategies were simulated to maximize cellulase production using glycerol to maintain a high cell concentration. The first simulation used a discrete feed and the second used a continuous feed of cellulose. The mathematical model proposed allows maintaining a high cell concentration and the addition of optimal small amounts of the inducer substrate to prevent inhibition of enzyme production.

The use of diesel fuel in crop and transportation operations is responsible for one third of the carbon emissions in sugarcane biorefineries. A possible solution is to replace it with biodiesel from lipids, directly produced from sugarcane by highly productive heterotrophic microalgae. In this study a heterotrophic microalgae biodiesel plant, integrated with a typical Brazilian sugarcane bio-refinery, was designed and evaluated. Molasses, steam, and electricity from sugarcane processing were used as inputs for microalgae production. For a non-integrated plant, the production cost of the microalgae biodiesel was estimated at 2.51 and 2.27 $/liter for fed-batch and continuous processes, respectively. Equipment for cultivation and carbon sources was the highest cost affecting the financial feasibility of the proposed design. For the integrated plant, at present ethanol and biodiesel selling prices, the profitability would be lower than a first-generation sugarcane bio-refinery using fossil diesel fuel for its operations. However, the CO₂ emissions would be reduced by up to 50 000 × 10³ kg per year at a cost of $83 10⁻³ kg⁻¹ CO₂-eq. If carbon credits are considered, the process becomes economically profitable even at present fuel prices.

Pretreatment of lignocellulosic biomass is required for many biorefinery processes. Previous studies have described hydrolysis of hemicelluloses by using liquid hot water (LHW) pretreatment. We evaluated the effect of carbonic acid originating from pressurized carbon dioxide during LHW pretreatment of poplar. The conditions applied covered temperatures from 120 to 200 °C, pretreatment times from 5 to 240 min and pressures from 1.0 to 2.2 MPa CO₂ or N₂. The pressure and the type of gas (CO₂ or N₂) did not have an effect on production of acetic acid, which functioned as a marker of progress of biomass hydrolysis. Results suggested that the presence of carbonic acid in the process does not significantly contribute to acidification. Deacetylation of lignocellulosic biomass can be achieved by LHW pretreatment irrespective of pressure and of gas type used, at the conditions tested.

Sesquiterpenes are a group of versatile, 15-carbon molecules with applications ranging from fuels to fine chemicals and pharmaceuticals. When produced by microbial fermentation at laboratory scale, solvents are often employed for reducing product evaporation and enhancing recovery. However, it is not clear whether this approach constitutes a favorable techno-economic alternative at production scale. In this study empirical correlations, mass transfer and process flow sheeting models were used to perform a techno-economic assessment of solvent-based processes at scales typical for flavors and fragrances (25 MT year⁻¹) and the fuel market (25 000 MT year⁻¹). Different solvent-based process options were compared to the current state of the art, which employs surfactants for product recovery. The use of solvents did reduce the sesquiterpene evaporation rate during fermentation and improved product recovery but it resulted in costs that were higher than, or similar to, the base case due to the additional equipment cost for solvent-product separation. However, when selecting solvents compatible with the final product formulation (e.g. in a kerosene enrichment process), unit costs as low as $0.7 kg⁻¹ can be achieved while decreasing environmental impact.

Biobased 2-butanol offers high potential as biofuel, but its toxicity toward microbial hosts calls for efficient techniques to alleviate product inhibition in fermentation processes. Aiming at the selective recovery of 2-butanol, the feasibility of a process combining in situ vacuum stripping followed by vapor adsorption has been assessed using mimicked fermentation media. The experimental vacuum stripping of model solutions and corn stover hydrolysate closely aligned with mass transfer model predictions. However, the presence of lignocellulosic impurities affected 2-butanol recovery yields resulting from vapor condensation, which decreased from 96 wt % in model solutions to 40 wt % using hydrolysate. For the selective recovery of 2-butanol from a vapor mixture enriched in water and carbon dioxide, silicalite materials were the most efficient, particularly at low alcohol partial pressures. Integrating in situ vacuum stripping with vapor adsorption using HiSiv3000 proved useful to effectively concentrate 2-butanol above its azeotropic composition (>68 wt %), facilitating further product purification.