Methane biohydroxylation into methanol by Methylosinus trichosporium OB3b: possible limitations and formate use during reaction.

Methane (CH4) hydroxylation into methanol (MeOH) by methanotrophic bacteria is an attractive and sustainable approach to producing MeOH. The model strain Methylosinus trichosporium OB3b has been reported to be an efficient hydroxylating biocatalyst. Previous works have shown that regardless of the bioreactor design or operation mode, MeOH concentration reaches a threshold after a few hours, but there are no investigations into the reasons behind this phenomenon. The present work entails monitoring both MeOH and formate concentrations during CH4 hydroxylation, where neither a gaseous substrate nor nutrient shortage was evidenced. Under the assayed reaction conditions, bacterial stress was shown to occur, but methanol was not responsible for this. Formate addition was necessary to start MeOH production. Nuclear magnetic resonance analyses with 13C-formate proved that the formate was instrumental in regenerating NADH; formate was exhausted during the reaction, but increased quantities of formate were unable to prevent MeOH production stop. The formate mass balance showed that the formate-to-methanol yield was around 50%, suggesting a cell regulation phenomenon. Hence, this study presents the possible physiological causes that need to be investigated further. Finally, to the best of our knowledge, this study shows that the reaction can be achieved in the native bacterial culture (i.e., culture medium containing added methanol dehydrogenase inhibitors) by avoiding the centrifugation steps while limiting the hands-on time and water consumption.

Experimental and modeling of tetracycline degradation in water in a flow-through enzymatic monolithic reactor.

In this work, the laccase from Trametes versicolor was immobilized in highly porous silica monoliths (0.6-cm diameter, 0.5-cm length). These monoliths feature a unique homogeneous network of interconnected macropores (20 µm) with mesopores (20 nm) in the skeleton and a high specific surface area (330 m2/g). The enzymatic monoliths were applied to degrade tetracycline (TC) in model aqueous solutions (20 ppm). For this purpose, a tubular flow-through reactor (FTR) configuration with recycling was built. The TC degradation was improved with oxygen saturation, presence of degradation products, and recirculation rate. The TC depletion reaches 50% in the FTR and 90% in a stirred tank reactor (CSTR) using crushed monoliths. These results indicate the importance of maintaining a high co-substrate concentration near active sites. A model coupling mass transfers with a Michaelis-Menten kinetics was applied to simulate the TC degradation in real wastewaters at actual TC concentration (2.8 10-4 ppm). Simulation results show that industrial scale FTR reactor should be suitable to degrade 90% of TC in 5 h at a flow rate of 1 mL/min in a single passage flow configuration. Nevertheless, the process could certainly be further optimized in terms of laccase activity, oxygen supply near active sites, and contact time.

A novel process for the covalent immobilization of laccases on silica gel and its application for the elimination of pharmaceutical micropollutants.

In the present work, pharmaceutical micropollutant degradation by laccase immobilized on silica through an innovative process is proposed. The influence of different parameters on the immobilization conditions was evaluated by a 23 full factorial design, and parameters leading to the highest activity were identified. Under these conditions, laccase activity reached 14 ± 2 U g-1 of silica with a protein immobilization yield of 35%. The biocatalyst characterization did not show any change in pH and thermal stabilities but enhanced the long-term storage of laccases. Immobilized T. versicolor laccases were then tested to remove four pharmaceutical micropollutants (amoxicillin, ciprofloxacin, carbamazepine, and sulfamethoxazole) in the presence of redox mediators (syringaldehyde, p-coumaric acid, and ABTS). High removal yields (50-100% according to the pollutant) were obtained within 4 h of treatment due to the synergistic effect of laccase-mediator biotransformation and adsorption on the support. Overall, the pharmaceuticals' removal efficiency was highly influenced by their physicochemical properties; however, the presence of redox mediators impacted not only the oxidation mechanism but also the interactions between the biocatalyst and micropollutants. Finally, the reusability of the biocatalyst was proved during 7 degradation cycles.

Spent brewer's yeast as a source of high added value molecules: a systematic review on its characteristics, processing and potential applications.

Development of new strategies to add-value to agro-industrial by-products are of environmental and economical importance. Innovative and low-cost sources of protein and bioactive peptides have been explored worldwide. Spent brewer's yeast (SBY) is the second most relevant by-product from the brewing industry, and despite its nutritional (about 50% protein, dry weight) and technological potential, it is still underused or needs to be disposed of. SBY cells need to be disrupted to release intracellular and cell wall proteins. This procedure has been performed using autolysis, glass bead milling, enzymatic hydrolysis and ultrasound processing. Enzymatic treatment is usually performed without prior purification and is a challenging process, which involves multiple factors, but has been successfully used as a strategy to add value to agro-industrial by-products. Scope and approach: in this review, we particularly focused on enzymatic hydrolysis as a strategy to promote SBY valorisation, illustrating the state-of-the-art processes used to produce protein extracts from this material as well as exploring fundamental concepts related to the particularities of yeast cell disruption and protein hydrolysis. Furthermore, innovative applications of value-added yeast by-products in food, biotechnological and pharmaceutical industries are presented and discussed. Key findings and conclusions: the discovery of valuable compounds found in spent yeasts as well as the development of new processing methodologies have been widening the possibilities of reuse and transformation of SBY as an ingredient and innovative matrix. Once released, yeast proteins and peptides may be applied as an innovative non-animal protein source or a functional and bioactive ingredient.

Alcoholic fermentation as a potential tool for coffee pulp detoxification and reuse: Analysis of phenolic composition and caffeine content by HPLC-DAD-MS/MS.

High-performance liquid chromatography with diode array (HPLC-DAD) and liquid chromatograph triple quadrupole mass spectrometry (HPLC-MS/MS) were used to characterize raw and fermented coffee pulps in terms of their phenolic composition and caffeine content. The qualitative analysis showed no significant differences between the raw and the fermented pulps. Free hydroxycinnamic acids (HAs) were mainly chlorogenic acids, with 5-caffeoylquinic acid as the major compound. Bound HAs released caffeic acids during alkaline hydrolysis, and no bound ferulic and p-coumaric acids were detected. The fermentation process allowed the detoxification of the pulp from caffeine by 50%, while significantly reducing the amounts of residue by 64%. Moreover, the fermented products could be further processed to provide high added-value molecules with potential industrial applications, providing a new source of income for the small coffee producers.

Membrane Fractionation of Protein Hydrolysates from By-Products: Recovery of Valuable Compounds from Spent Yeasts.

Spent brewer's yeast (Saccharomyces sp.), the second most generated by-product from the brewing industry, contains bioactive and nutritional compounds with high added value such as proteins (40-50%), polysaccharides, fibers and vitamins. Molecules of interest from agro-industrial by-products need to be extracted, separated, concentrated, and/or purified so that a minimum purity level is achieved, allowing its application. Enzymatic hydrolysis has been successfully used in the production of peptides and protein hydrolysates. The obtained hydrolysates require efficient downstream processes such as membrane technology, which is an important tool for the recovery of thermolabile and sensitive compounds from complex mixtures, with low energy consumption and high specificity. The integration of membrane techniques that promote the separation through sieving and charge-based mechanisms is of great interest to improve the purity of the recovered fractions. This review is specifically addressed to the application of membrane technologies for the recovery of peptides from yeast protein hydrolysates. Fundamental concepts and practical aspects relative to the ultrafiltration of agro-industrial protein hydrolysates will be described. Challenges and perspectives involving the recovery of peptides from yeast protein hydrolysates will be presented and thoroughly discussed.

Potentialities and Limits of Some Non-thermal Technologies to Improve Sustainability of Food Processing.

In the whole food production chain, from the farm to the fork, food manufacturing steps have a large environmental impact. Despite significant efforts made to optimize heat recovery or water consumption, conventional food processing remains poorly efficient in terms of energy requirements and waste management. Therefore, in the few last decades, much research has focused on the development of alternative non-thermal technologies. Some of them, such as membrane separation processes, hydrostatic or dynamic high pressure, dense phase or high-pressure carbon dioxide, and pulsed electric fields (PEFs) have been extensively studied for cold pasteurization, concentration, extraction, or food functionalization. However, it is still difficult to evaluate the actual advantages or limits of these innovative processing technologies to replace conventional processes. Thus, the overall aim of this paper is to present an overview of the most relevant studies dealing with the potentialities and limits of these non-thermal technologies to improve sustainability of food processing. After a brief presentation of the physical principles of these technologies, the paper illustrates how these technologies could play a decisive role for sustainable food preservation or valorization of raw materials and by-products.

Potentialities of active membranes with immobilized laccase for Bisphenol A degradation.

Herein, we report the development of immobilized laccase based membrane bioreactor as a novel bio-catalytic system for the degradation of emerging endocrine disruptor i.e., Bisphenol A. Two laccase forms i.e. (1) in-house isolated and purified from an indigenous white-rot fungi Pycnoporus sanguineus (CS43) and (2) Trametes versicolor (commercial laccase from Sigma-Aldrich®) were immobilized on a multi-channel ceramic membrane (1.4µm in diameter) using 4% glutaraldehyde as a cross-linking agent. The immobilization yield and bisphenol A degradation activities of immobilized laccases were recorded at various pH levels. The surface topographies of immobilized-laccase membranes were accessed by scanning electron microscopy. In this study, 100% degradation of bisphenol A (20mg/L) was achieved in less than 24h in the presence of laccase from P. sanguineus (CS43) (620.55±14.85U/L) and T. versicolor (620.55±14.85U/L). The enzymes showed an optimal activity at pH 5 and 5.4 with a degradation rate of 204.8±1.8 and 79.0±0.1µmol/min/U for P. sanguineus (CS43) and T. versicolor, respectively. In conclusion, the highest immobilization of unit per square centimeter and efficient degradation potentiality strongly recommend the newly developed immobilized laccase based membrane bioreactor as a novel tool to tackle emerging contaminants degradation issues.

Removal of antibiotics in wastewater by enzymatic treatment with fungal laccase - Degradation of compounds does not always eliminate toxicity.

In this study, the performance of immobilised laccase (Trametes versicolor) was investigated in combination with the mediator syringaldehyde (SYR) in removing a mixture of 38 antibiotics in an enzymatic membrane reactor (EMR). Antibiotics were spiked in osmosed water at concentrations of 10µg·L(-1) each. Laccase without mediator did not reduce the load of antibiotics significantly. The addition of SYR enhanced the removal: out of the 38 antibiotics, 32 were degraded by >50% after 24h. In addition to chemical analysis, the samples' toxicity was evaluated in two bioassays (a growth inhibition assay and the Microtox assay). Here, the addition of SYR resulted in a time-dependent increase of toxicity in both bioassays. In cooperation with SYR, laccase effectively removes a broad range of antibiotics. However, this enhanced degradation induces unspecific toxicity. If this issue is resolved, enzymatic treatment may be a valuable addition to existing water treatment technologies.

Alkane biohydroxylation: Interests, constraints and future developments.

Alkanes constitute one of the vastest reserves of raw materials for the production of fine chemicals. This paper focuses on recent advances in alkane biohydroxylation, i.e. the bioactivation of alkanes into their corresponding alcohols. Enzyme and whole-cell biocatalysts have been reviewed. Process considerations to implement such biocatalysts in bioreactors at large scale by coupling the bioconversion with cofactor regeneration and product removal are also discussed.

Potentialities of a membrane reactor with laccase grafted membranes for the enzymatic degradation of phenolic compounds in water.

This paper describes the degradation of phenolic compounds by laccases from Trametes versicolor in an enzymatic membrane reactor (EMR). The enzymatic membranes were prepared by grafting laccase on a gelatine layer previously deposited onto α-alumina tubular membranes. The 2,6-dimethoxyphenol (DMP) was selected  from among the three different phenolic compounds tested (guaiacol, 4-chlorophenol and DMP) to study the performance of the EMR in dead end configuration. At the lowest feed substrate concentration tested (100 mg·L-1), consumption increased with flux (up to 7.9 × 103 mg·h-1·m-2 at 128 L·h-1·m-2), whereas at the highest substrate concentration (500 mg·L-1), it was shown that the reaction was limited by the oxygen content.

Membrane bioprocesses for pharmaceutical micropollutant removal from waters.

The purpose of this review work is to give an overview of the research reported on bioprocesses for the treatment of domestic or industrial wastewaters (WW) containing pharmaceuticals. Conventional WW treatment technologies are not efficient enough to completely remove all pharmaceuticals from water. Indeed, these compounds are becoming an actual public health problem, because they are more and more present in underground and even in potable waters. Different types of bioprocesses are described in this work: from classical activated sludge systems, which allow the depletion of pharmaceuticals by bio-degradation and adsorption, to enzymatic reactions, which are more focused on the treatment of WW containing a relatively high content of pharmaceuticals and less organic carbon pollution than classical WW. Different aspects concerning the advantages of membrane bioreactors for pharmaceuticals removal are discussed, as well as the more recent studies on enzymatic membrane reactors to the depletion of these recalcitrant compounds.

Enzymatic membrane reactor for full saccharification of ionic liquid-pretreated microcrystalline cellulose.

Ultrafiltration reactors based on polymeric or ceramic membranes were shown to be suitable catalytic systems for fast enzymatic saccharification of cellulose, allowing the full recovery and reuse of enzymes. By pre-treating cellulose with the IL 1-butyl-3-methylimidazolium chloride, the suitability of this substrate for enzymatic saccharification in a reactor based on polymeric ultrafiltration membranes was demonstrated, leading to 95% cellulose hydrolysis in 4h at 50°C. The filtration process gave a clear glucose solution (up to 113 mM) at constant permeate flow (24.7 L h(-1) m(-2)), allowing the enzyme to be reused for 9 operation cycles under semi-continuous operation, without any loss of enzyme activity. Under continuous operation mode and using ceramic ultrafiltration membranes at different residence times, the enzymatic reactor showed constant profiles in both the permeate flow rate and the glucose concentration, demonstrating the excellent suitability of the proposed approach for the saccharification of cellulose.

Analysis of the main components of the aguamiel produced by the maguey-pulquero (Agave mapisaga) throughout the harvest period.

The main characteristics of the aguamiel (maguey-pulquero sap) during the harvest period of the Agave mapisaga plants were assessed to establish its stability through time and the industrial potential of its components. Only minor differences in aguamiel composition were detected among samples collected at different time points of the harvest period. The aguamiel analyzed contained 11.5 wt % of dry matter, which was composed mainly of sugars (75 wt %). Among these sugars, 10 wt % were fructo-oligosaccharides (FOS), which are known to be important in the food industry for their prebiotic properties. Other components include 0.3 wt % of free amino acids (with most essential amino acids and four neurotransmitters: GABA, GLY, GLX, and ASX), 3 wt % of proteins, and 3 wt % of ashes.

Membrane engineering in biotechnology: quo vamus?

Membranes are essential to a range of applications, including the production of potable water, energy generation, tissue repair, pharmaceutical production, food packaging, and the separations needed for the manufacture of chemicals, electronics and a range of other products. Therefore, they are considered to be "dominant technologies" by governments and industry in several prominent countries--for example, USA, Japan and China. When combined with catalysts, membranes are at the basis of life, and membrane-based biomimetism is a key tool to obtain better quality products and environmentally friendly developments for our societies. Biology has a main part in this global landscape because it simultaneously provides the "model" (with natural biological membranes) and represents a considerable field of applications for new artificial membranes (biotreatments, bioconversions and artificial organs). In this article, our objective is to open up this enthralling area and to give our views about the future of membranes in biotechnology.