Structural alteration of cocoa bean shell fibers through biological treatment using Penicillium roqueforti.

Lignocellulosic residues, such as cocoa bean shell (FI), are generated in large quantities during agro-industrial activities. Proper management of residual biomass through solid state fermentation (SSF) can be effective in obtaining value-added products. The hypothesis of the present work is that the bioprocess promoted by P. roqueforti can lead to structural changes in the fibers of the fermented cocoa bean shell (FF) that confer characteristics of industrial interest. To unveil such changes, the techniques of FTIR, SEM, XRD, TGA/TG were used. After SSF, an increase of 36.6% in the crystallinity index was observed, reflecting the reduction of amorphous components such as lignin in the FI residue. Furthermore, an increase in porosity was observed through the reduction of the 2θ angle, which gives the FF a potential candidate for applications of porous products. The FTIR results confirm the reduction in hemicellulose content after SSF. The thermal and thermogravimetric tests showed an increase in the hydrophilicity and thermal stability of FF (15% decomposition) in relation to the by-product FI (40% decomposition). These data provided important information regarding changes in the crystallinity of the residue, existing functional groups and changes in degradation temperatures.

This work presents a new approach for solid state fermentation based on the study of structural changes caused by Penicillium roquefort, which is important to understand the changes in the lignocellulosic matrix after the fungus growth. The results provided important information regarding changes in the crystallinity of the residue, existing functional groups and changes in degradation temperatures. Consequently, they can help in proposals for the total use of the residual solid after fermentation, as well as contribute to reducing the lack of this information in the literature.

Reduction of scum accumulation through the addition of low-cost enzymatic extract in the feeding of high-rate anaerobic reactor.

This work evaluates the reduction of scum accumulation on the top surface of upflow anaerobic sludge blanket (UASB) reactors by the addition of hydrolytic enzymes in their feed. For over 1 year, two UASB reactors of 1.4 L were maintained at 30 °C and continuously fed with synthetic domestic wastewater (containing 150 mg/L of soybean oil) under a hydraulic retention time of 10 h. The Control reactor was only fed with synthetic wastewater. Beginning at the 226th day of operation, low-cost hydrolytic enzymes (obtained by solid-state fermentation of Aspergillus terreus, a fungus isolated from a primary sewage sludge) were added into the feed of the other reactor (Test) for a lipase activity of 24 U/L, considerably reducing the formation of scum. In the Test reactor, the scum showed oil and grease (O&G) concentration between 0.8 and 1.3 g/L and an accumulation rate of 20 to 27 mg O&G/d. In the Control reactor, the scum had values twice as high (1.5-2.5 g/L and 34-51 mg O&G/d, respectively) and there were more operational problems. During the entire period of operation, both reactors presented high chemical oxygen demand removal (>80%), with no loss of effluent quality due to the addition of the enzymes.

Studying the expression of a lipase from Pyrococcus furiosus using response surfaces.

The need to find more stable catalysts has encouraged the study of naturally resilient enzymes, such as those found in extremophile organisms. In the present work, the influence of rare codons on the expression in Escherichia coli of the lipase Pf2001Δ60 from Pyrococcus furiosus was evaluated. Expression was carried out in two E. coli strains, BL21(DE3)pLysS and the rare tRNA supplemented Rosetta(DE3)pLysS. 3(2) factorial design was used to appraise the influence of temperature and inducer concentration on enzyme expression every hour for the 4-h expression period. Four response surfaces were constructed for each time, and the statistical parameters were evaluated. Lipase production was twice as high for Rosetta(DE3)pLysS than for BL21(DE3)pLysS. The factorial design indicated that optimal expression occurred at 30 °C after 4h, with lipase production of 240 U/L. The analysis of statistical parameters during the expression time showed that the velocity at which the enzyme was produced affected cell growth and maximum activity, with a higher speed leading to lower expression and cell growth. The presence of rare tRNAs prevented bottlenecks in lipase expression, and the experimental design was shown to be important for maximizing the production strategies and minimizing the metabolic load to which the host is subjected.

Simultaneous lipase production and immobilization: morphology and physiology study of Penicillium simplicissimum in submerged and solid-state fermentation with polypropylene as an inert support.

The influence of different carbon sources (glucose (G), olive oil (O), and a combination of both (GO)) in the physiology (biomass and lipase production) and morphology (light and environmental and scanning electron microscopy) of the fungus Penicillium simplicissimum by applying submerged (SmF) and solid-state (SSF) fermentations was investigated. The cultivation was carried out using polypropylene as hydrophobic inert support in SmF and SSF to understand better the influence of a support for the fungus growth and also provides the immobilization of lipases during its production. Micrographs show different morphologies: in SSF, the fungus grows on and inside the inert support independent of the media; in SmF, the formation of high-density spherical pellets obtained in medium GO leads to the best productivity and specific product yield Yp/x..Conidiation is observed mainly in SSF, a few in SmF with polypropylene as inert support and not in SmF, which may indicate a stress condition in SSF. Possibly, the morphology acquired by the fungus under stressful conditions may be the key to the higher biomass and lipase productivity at SSF. The developed process with simultaneous production and immobilization of lipase leads to a new promissory biocatalyst once it can be directly applied with no need for downstream processes.

Effect of the solid state fermentation of cocoa shell on the secondary metabolites, antioxidant activity, and fatty acids.

During cocoa (Theobroma cacao L.) processing, the accumulated cocoa shell can be used for bioconversion to obtain valuable compounds. Here, we evaluate the effect of solid-state fermentation of cacao flour with Penicillium roqueforti on secondary metabolite composition, phenol, carotenoid, anthocyanin, flavonol, and fatty acids contents, and antioxidant activity. We found that the total concentrations of anthocyanins and flavonols did not change significantly after fermentation and the phenolic compound and total carotenoid concentrations were higher. The fermentation process produced an increase in saponin concentration and antioxidant activity, as well as significant changes in the levels of oleic, linoleic, gamma-linolenic, and saturated fatty acids. Based on our findings, we propose that the reuse of food residues through solid state fermentation is viable and useful.

Surface imaging of the filamentous fungus Penicillium simplicissimum growing in a solid-state fermentation system.

A comparative study on the lipase-producing fungus Penicillium simplissicimum, grown on a tray type solid-state fermentation (SSF) bioreactor, was performed using stereoscopy, focus Z-stacking stereoscopic images, field emission scanning electron microscopy (FE-SEM) and environmental scanning electron microscopy (ESEM) to better characterize the morphology of filamentous fungi in SSF and their distribution over the solid matrix. The imaging of live fungal samples using a stereomicroscope with focus Z-stacking showed differences in colonization between a static SSF and an intermittent agitated SSF. A comparison of the stereomicroscopy, hi-vacuum and environmental scanning electron microscopy results obtained using different protocols for sample processing showed that fixation with osmium tetroxide vapor and subsequent hydrated imaging is the best combination of sample preparation and imaging conditions for keeping the arrangement of the aerial hyphae and conidia morphology closer to the natural state. These combined methodologies can be applied in the SSF of fungal growth to characterize the formation of conidiophores over time, the conidia morphology and the spatial organization after their release from conidiophores. Mycelium colonization over the matrix, which is an important characteristic related to the production of different biotechnological products, could be observed and provide more knowledge about fungal physiology behavior during SSF.

Lipase production and Penicillium simplicissimum morphology in solid-state and submerged fermentations.

A comparative study of Penicillium simplicissimum morphology and lipase production was performed using solid-state (SSF) and submerged (SmF) fermentation. SSF was carried out on babassu cake as culture medium and SmF on a semi-synthetic medium and a medium based on suspended babassu cake grains. Yield of product on biomass, specific activity and conidia production were 3.3-, 1.3- and 2-fold higher in SSF. In SmF, the type of fungus growth differed according to the medium. Using the semi-synthetic medium, the fungus formed densely interwoven mycelial masses without conidia production, whereas using the babassu-based medium the fungus formed free mycelia and adhered to the surfaces of the grains, producing conidia. The results show that babassu cake induces conidiation in SmF. In SSF, the fungus not only grew on the surface of the grains, producing conidia abundantly, but also effectively colonized and penetrated the babassu particles. The high conidia production and lipase productivity in SSF may be related to the low availability of nutrients or to other stimuli associated with this type of fermentation. Thus, the high production of the thermostable P. simplicissimum lipase, using a non-supplemented, low-cost agro-industrial residue as the culture medium, demonstrates the biotechnological potential of SSF for the production of industrial enzymes.

Lipase production by solid-state fermentation in fixed-bed bioreactors

O fungo Penicillium simplicissimum se mostrou, em trabalhos anteriores, um ótimo produtor de lipase por fermentação no estado sólido, quando cultivado em biorreatores do tipo bandeja, utilizando a torta de babaçu como meio de cultura. Com o objetivo de aumentar a produtividade e possibilitar uma ampliação de escala, foi investigado, no presente trabalho, o emprego de biorreatores de leito fixo com aeração forçada. Os biorreatores utilizados tinham 4 cm de diâmetro interno e 14 cm de altura útil. Empregando-se planejamento estatístico de experimentos como ferramenta, foram avaliadas as influências da temperatura e da vazão de ar sobre a produção de lipase nestes biorreatores. Os resultados obtidos permitiram ajustar um modelo empírico, o qual indicou que maiores atividades lipásicas são alcançadas para temperaturas mais baixas e vazões de ar mais altas. A atividade lipásica máxima (26,4 U/g) foi obtida para temperatura de 27°C e vazão de ar de 0,8 L/min.