An overview on fermentation strategies to overcome lignocellulosic inhibitors in second-generation ethanol production using cell immobilization.

The development of technologies to ferment carbohydrates (mainly glucose and xylose) obtained from the hydrolysis of lignocellulosic biomass for the production of second-generation ethanol (2G ethanol) has many economic and environmental advantages. The pretreatment step of this biomass is industrially performed mainly by steam explosion with diluted sulfuric acid and generates hydrolysates that contain inhibitory compounds for the metabolism of microorganisms, harming the next step of ethanol production. The main inhibitors are: organic acids, furan, and phenolics. Several strategies can be applied to decrease the action of these compounds in microorganisms, such as cell immobilization. Based on data published in the literature, this overview will address the relevant aspects of cell immobilization for the production of 2G ethanol, aiming to evaluate this method as a strategy for protecting microorganisms against inhibitors in different modes of operation for fermentation. This is the first overview to date that shows the relation between inhibitors, cells immobilization, and fermentation operation modes for 2G ethanol. In this sense, the state of the art regarding the main inhibitors in 2G ethanol and the most applied techniques for cell immobilization, besides batch, repeated batch and continuous fermentation using immobilized cells, in addition to co-culture immobilization and co-immobilization of enzymes, are presented in this work.

Nanoflowers: A New Approach of Enzyme Immobilization.

Enzymes are biocatalysts known for versatility, selectivity, and brand operating conditions compared to chemical catalysts. However, there are limitations to their large-scale application, such as the high costs of enzymes and their low stability under extreme reaction conditions. Immobilization techniques can efficiently solve these problems; nevertheless, most current methods lead to a significant loss of enzymatic activity and require several steps of activation and functionalization of the supports. In this context, a new form of immobilization has been studied: forming organic-inorganic hybrids between metal phosphates as inorganic parts and enzymes as organic parts. Compared to traditional immobilization methods, the advantages of these nanomaterials are high surface area, simplicity of synthesis, high stability, and catalytic activity. The current study presents an overview of organic-inorganic hybrid nanoflowers and their applications in enzymatic catalysis.

A Perspective Review on the Application of Polyacrylonitrile-Based Supports for Laccase Immobilization.

The use of laccases applied in bioremediation processes has been increasingly studied, given the urgent need to overcome the environmental problems caused by emerging contaminants. It is known that immobilized enzymes have better operational stability under reaction conditions, allowing for greater applicability. However, given the lack of commercially available immobilized laccases, the search for immobilization materials and methods continues to gain effort. The use of polyacrylonitrile (PAN) to immobilize enzymes has been investigated since it is a low-cost material and can be modified and functionalized to well interact with the enzyme. This polymer can be used with different morphologies such as fibers, beads, and core-shell, presenting as an easily applicable alternative. This review presents the missing link between polymer and enzyme through an overview of PAN's current use as support for laccase immobilization and polymer functionalization methods, considering the importance of immobilized laccases in several industrial sectors.

Production of Pleurotus sajor-caju crude enzyme broth and its applicability for the removal of bisphenol A.

Bisphenol A is an endocrine interfering compound, produced and used on a large scale worldwide. Chemical and biologic methods can be used to remove it from the environment. Biological methods are considered less costly, safer and, according to green chemistry definitions, an environmentally correct method. Considering the use of a crude enzyme broth, without any downstream process, the costs could be mostly reduced. Thus, the removal of bisphenol A by Pleurotus sajor-caju crude enzyme broth was investigated. Initially, the agro-industrial wastes were characterized and, the composition of the culture medium and the bioreactor culture conditions were defined. The enzyme produced in the highest concentration was characterized and the crude broth used in the bisphenol A removal assays. The OXI45 culture medium presented the highest laccase activity (1,850.7 U L-1, 350 rpm). Greater laccase stability was observed at 20 - 40 oC and pHs 5 - 7. Vanillin and ferulic acid (considered mediator compounds) were identified in the crude broth, probably helping on the obtention of the high value of removal effectiveness (0.052 mg U-1 h-1). The results indicate the potential use of the Pleurotus sajor-caju crude enzyme broth to obtain an enzymatic formulation for application in the environmental area.

Practical and Rapid Membrane-Based Biosensor for Phenol Using Copper/Calcium-Enzyme Hybrid Nanoflowers.

Phenol, a pollutant frequently found in chemical industries effluents, is highly toxic even in low concentrations. This study reports a green, simple, and rapid method for qualitative phenol biosensing using horseradish peroxidase (HRP) hybrid nanoflowers made with copper (Cu2+-hNF) or calcium (Ca2+-hNF) ions. The enzyme was immobilized through protein-inorganic self-assembly into hybrid structures and subsequently supported onto a polyvinylidene fluoride (PVDF) membrane. SEM, EDS, FTIR, and XRD techniques sustained the effective enzyme encapsulation into hybrid structures. The protein concentration in the structures was 0.25 mg.mL-1 for both ions. The best temperature and pH were 60 °C and 7.4, respectively, for both hybrids and the free enzyme, suggesting that the immobilization did not affect the optimal conditions of the free HRP. Thermal stability from 25 to 70 °C and pH stability from 4.0 to 9.0 of the hybrids were also determined. Finally, using copper and calcium hybrids, both biosensors produced onto a PVDF membrane could detect phenol in concentrations ranging from 0.72 to 24.00 µmol.mL-1 in 1 min. In contrast, control biosensors produced with free enzyme have not presented a visible color change in the same conditions. The findings suggest a promising application of the developed biosensors in functional phenol detection.

A model for growth of a single fungal hypha based on well-mixed tanks in series: simulation of nutrient and vesicle transport in aerial reproductive hyphae.

Current models that describe the extension of fungal hyphae and development of a mycelium either do not describe the role of vesicles in hyphal extension or do not correctly describe the experimentally observed profile for distribution of vesicles along the hypha. The present work uses the n-tanks-in-series approach to develop a model for hyphal extension that describes the intracellular transport of nutrient to a sub-apical zone where vesicles are formed and then transported to the tip, where tip extension occurs. The model was calibrated using experimental data from the literature for the extension of reproductive aerial hyphae of three different fungi, and was able to describe different profiles involving acceleration and deceleration of the extension rate. A sensitivity analysis showed that the supply of nutrient to the sub-apical vesicle-producing zone is a key factor influencing the rate of extension of the hypha. Although this model was used to describe the extension of a single reproductive aerial hypha, the use of the n-tanks-in-series approach to representing the hypha means that the model has the flexibility to be extended to describe the growth of other types of hyphae and the branching of hyphae to form a complete mycelium.

Kinetic and stoichiometric parameters in the production of carotenoids by Sporidiobolus salmonicolor (CBS 2636) in synthetic and agroindustrial media.

With the objective of determining the kinetic behavior (growth, substrate, pH, and carotenoid production) and obtain the stoichiometric parameters of the fermentative process by Sporidiobolus salmonicolor in synthetic and agroindustrial media, fermentations were carried out in shaken flasks at 25 degrees C, 180 rpm, and initial pH of 4.0 for 120 h in the dark, sampling every 6 h. The maximum concentrations of total carotenoids in synthetic (913 microg/L) and agroindustrial (502 microg/L) media were attained approximately 100 h after the start of the fermentative process. Carotenoid bioproduction is associated with cell growth and the ratio between carotenoid production and cell growth (Y (P/X)) is 176 and 163 microg/g in the synthetic and agroindustrial media, respectively. The pH of the agroindustrial fermentation medium varied from 4.2 to 8.5 during the fermentation. The specific growth rate (micro (X)) for S. salmonicolor in synthetic and agroindustrial media was 0.07 and 0.04 h(-1), respectively. The synthetic medium allowed for greater productivity, obtaining maximum cell productivity (P (x)) of 0.08 g L(-1) h(-1) and maximum total carotenoid productivity (P (car)) of 14.2 microg L(-1) h(-1). Knowledge of the kinetics of a fermentative process is of extreme importance when transposing a laboratory experiment to an industrial scale, as well as making a quantitative comparison between different culture conditions.

Production of bio-inseticide Bacillus thuringiensis var. israelensis in semicontinuous processes combined with batch processes for sporulation

The aim of this work was to study the production of the bioinsecticide using the aerobic cultivation. Two-stage cultivation process was used: aerobic vegetative growth and end-phase of sporulation (with aeration and without aeration). The cuts of working volume evaluated were 20, 40, 60 and 80 percent. The highest value of spore concentration (2.17x10(9) CFU/mL) and spore productivity (7.2x10(7) CFU/mL.h) were observed using SP60. The lethal concentration (CL50) obtained through the aeration process and non-aeration process were 0.21 and 0.05 mg/L, respectively. The specific potential of the bioinsecticide produced without aeration (0.025 mg/10-12 CFU) was two times higher than that obtained with aeration (0.05 mg/10-12 CFU).

Anaerobic biodegradability of dairy wastewater pretreated with porcine pancreas pipase

Lipids-rich wastewater was partial hydrolyzed with porcine pancreas lipase and the efficiency of the enzymatic pretreatment was verified by the comparative biodegradability tests (crude and treated wastewater). Alternatively, simultaneous run was carried out in which hydrolysis and digestion was performed in the same reactor. Wastewater from dairy industries and low cost lipase preparation at two concentrations (0.05 and 0.5 percent w.v-1) were used. All the samples pretreated with enzyme showed a positive effect on organic matter removal (Chemical Oxygen Demand-COD) and formation of methane. The best results were obtained when hydrolysis and biodegradation were performed simultaneously, attaining high COD and color removal independent of the lipase concentration. The enzymatic treatment considerably improved the anaerobic operational conditions and the effluent quality (lower content of suspended solids and less turbidity). Thus, the use of enzymes such as lipase seemed to be a very promising alternative for treating the wastewaters having high fat and grease contents, such as those from the dairy industry.

O presente trabalho teve como objetivo o pré-tratamento de efluente da indústria de laticínios na hidrólise de lipídeos, empregando lipase de fonte de células animais de baixo custo disponível comercialmente (pâncreas de porco) na formação de gás metano por biodegradabilidade anaeróbia empregando diferentes concentrações de lipase (0,05 e 0,5 por cento w.v-1). A utilização de lipase no pré-tratamento do efluente acelerou a hidrólise de lipídeos e, conseqüentemente, auxiliou o tratamento biológico resultando na redução da matéria orgânica em termos de Demanda Química de Oxigênio (DQO), cor e sólidos em suspensão como lipídeos. Os melhores resultados em termos de remoção de DQO e cor foram obtidos quando a hidrólise e biodigestão foram realizadas simultaneamente, independente da concentração de lipase empregada. Estes resultados mostram que a hidrólise e biodigestão podem ser realizadas simultaneamente empregando baixa concentração de lipase (0,05 por cento w.v-1), contribuindo para a redução de custo e tempo no processo de biodegradação aneróbia.

Viability and infectivity of an ectomycorrhizal inoculum produced in an airlift bioreactor and immobilized in calcium alginate

The viability and infectivity of an ectomycorrhizal inoculum (isolate UFSC-Rh90, Rhizopogon nigrescens), produced by submerged cultivation in an airlift bioreactor and immobilized in beads of calcium alginate gel, was studied. Inoculum remained 100 percent viable after 18 months in a 0.85 percent NaCl solution at 8 ± 1°C. Mycelium grew from the beads after 48 h when they were placed on a solid culture medium at 25 ± 1°C. Viability of pellets of non-immobilized mycelium stored under the same conditions decreased gradually after the third month of storage, reaching 0 percent by the 12th month. These pellets presented a gradual darkening, which was more intense in those located near the surface of the NaCl solution. In culture medium, these dark pellets showed no viability. Gel immobilization helps to maintain mycelium viability during storage and offers a physical protection when the inoculum is applied to the planting substrate. After eight months refrigeration, the immobilized inoculum was still able to infect Pinus taeda seedlings, colonizing an average of 37 percent of the root tips when inoculated in the plant growth substrate under greenhouse conditions. This inoculum presents a commercial potential to be produced and applied in forest nurseries.

Estudou-se a viabilidade e a infectividade de inoculante fúngico ectomicorrízico (isolado UFSC-Rh90, Rhizopogon nigrescens), produzido através de cultivo submerso em biorreator airlift e encapsulado em gel de alginato de cálcio. O inoculante permaneceu viável após 18 meses em solução de NaCl (0,85 por cento) a 8 ± 1°C. O micélio emergiu dessas cápsulas após 48 h de incubação a 25 ± 1°C em meio de cultura sólido. A viabilidade dos pellets de micélio não imobilizado, armazenados sob as mesmas condições, reduziu-se gradualmente após três meses de armazenamento e atingiu 0 por cento aos 12 meses. Esses pellets apresentaram um escurecimento gradual que foi mais intenso naqueles localizados próximos à superfície da solução de NaCl. Em meio de cultura, os pellets escurecidos mostraram-se inviáveis. A imobilização em gel mantém a viabilidade do micélio durante o armazenamento, além de oferecer uma barreira física quando aplicado ao substrato de plantio. Após oito meses de armazenamento sob refrigeração, o inoculante imobilizado colonizou uma média de 37 por cento das raízes curtas de mudas de Pinustaeda, quando aplicado ao substrato de plantio sob condições de casa-de-vegetação. Esse inoculante apresenta potencial para produção comercial e aplicação nos viveiros florestais.