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.

Growth of Methylobacterium organophilum in Methanol for the Simultaneous Production of Single-Cell Protein and Metabolites of Interest.

Research background: This study aims to monitor the growth of the methylotrophic bacteria Methylobacterium organophilum in a culture medium with methanol as a carbon source and to verify the production of unicellular proteins and other biomolecules, such as carotenoids, exopolysaccharides and polyhydroxyalkanoates, making them more attractive as animal feed. Experimental approach: Bacterial growth was studied in shake flasks using different carbon/nitrogen (C:N) ratios to determine their best ratio for achieving the highest volumetric productivity of cells and substrate consumption rate. This optimal parameter was further used in a fed-batch operating bioreactor system to define the kinetic profile of cell growth. Methanol consumption was measured by HPLC analysis and the extracted pigments were analyzed by liquid chromatography/mass spectrometry. Chemical composition and rheological properties of the produced exopolysaccharides were also determined. Results and conclusions: The best experimental parameters were verified using an initial methanol concentration of 7 g/L in the culture medium. The same initial substrate concentration was used in the fed-batch operation and after 60 h of cultivation 5 g/L of biomass were obtained. The accumulation of carotenoids associated with cell growth was monitored, reaching a concentration of 1.6 mg/L at the end of the process. These pigments were then analyzed and characterized as a set of xanthophylls (oxidized carotenoids). In addition, two other product types were identified during the fed-batch operation: exopolysaccharides, which reached a concentration of 8.9 g/L at the end of the cultivation, and an intracellular granular structure that was detected by transmission electron microscopy (TEM), suggesting the accumulation of polyhydroxyalkanoate (PHA), most likely polyhydroxybutyrate. Novelty and scientific contribution: Methylobacterium organophilum demonstrated a unique ability to produce compounds of commercial interest. The distinct metabolic diversity of this bacterium makes room for its use in biorefineries.

Homoscleromorpha-derived Bacillus spp. as potential sources of biotechnologically-relevant hydrolases and biosurfactants.

Despite hydrolytic exoenzymes and biosurfactants having been gradually reported from the poriferan microbiome, little is known about these bioproducts in microorganisms inhabiting Homoscleromorpha sponges. Here, we investigated the production of hydrolases and biosurfactants in bacteria isolated from three shallow-water homoscleromorph species, Oscarella sp., Plakina cyanorosea, and Plakina cabofriense. A total of 99 of 107 sponge-associated bacterial isolates exhibited activity for at least one of the analyzed hydrolases. Following fermentation in Luria-Bertani (LB) and Tryptic Soy Broth (TSB), two isolates, 80BH11 and 80B1:1010b, showed higher lipase and peptidase activities. Both of them belonged to the Bacillus genus and were isolated from Oscarella. Central composite design leveraged up the peptidase activity in 280% by Bacillus sp. 80BH11 in the TSB medium for 48 h at 30 °C. The optimized model also revealed that pH 6.5 and 45 °C were the best conditions for peptidase reaction. In addition, Bacillus sp. 80BH11 was able to release highly emulsifying and remarkably stable surfactants in the LB medium. Surfactin was finally elucidated as the biosurfactant generated by this sponge-derived Bacillus. In conclusion, we hope to have set the scenery for further prospecting of industrial enzymes and biosurfactants in Homoscleromorpha microbiomes.

New method for rapid identification and quantification of fungal biomass using ergosterol autofluorescence.

This research reports on the development of a method to identify and quantify fungal biomass based on ergosterol autofluorescence using excitation-emission matrix (EEM) measurements. In the first stage of this work, several ergosterol extraction methods were evaluated by APCI-MS, where the ultrasound-assisted procedure showed the best results. Following an experimental design, various quantities of the dried mycelium of the fungus Schizophyllum commune were mixed with the starchy solid residue (BBR) from the babassu (Orbignya sp.) oil industry, and these samples were subjected to several ergosterol extraction methods. The EEM spectral data of the samples were subjected to Principal Component Analysis (PCA), which showed the possibility to qualitatively evaluate the presence of ergosterol in the samples by ergosterol autofluorescence without the addition of any reagent. In order to assess the feasibility of quantifying fungal biomass using ergosterol autofluorescence, the EEM spectral data and known amounts of fungal biomass were modeled using partial least squares (PLS) regression and a procedure of backward selection of predictors (AutoPLS) was applied to select the Excitation-Emission wavelength pairs that provide the lowest prediction error. The results revealed that the amount of fungal biomass in samples containing interfering substances (BBR) can be accurately predicted with R2CV = 0.939, R2P = 0.936, RPDcv = 4.07, RPDp = 4.06, RMSECV = 0.0731 and RMSEP = 0.0797. In order to obtain an easy-to-understand equation that expresses the relationship between fungal biomass and fluorescence intensity, multiple linear regression (MLR) was applied to the VIP variables selected by the AutoPLS method. The MLR model selected only 2 variables and showed a very good performance, with R2CV = 0.862, R2P = 0.809, RPDcv = 2.18, RPDp = 2.35, RMSECV = 0.137 and RMSEP = 0.138. This study demonstrated that ergosterol autofluorescence can be successfully used to quantify fungal biomass even when mixed with agroindustrial residues, in this case BBR.

A microplate assay for extracellular hydrolase detection.

This article presents a new qualitative method to detect enzyme activity replacing the conventional Agar-Petri dishes. This new method is a simple rapid and low-cost technique that uses 24-well microplates. The detection of hydrolases producing microorganisms in bioprospecting studies by qualitative methods is time consuming, costly and requires a large quantity of strains or enzymatic extracts. Tests with different substrate concentrations (0 to 20 g/L) in agar solution for the enzymatic hydrolysis analysis were performed to determine the best substrate concentrations in 24-well microplates. Other quantitative and analytical methods, such as enzymatic assays and thin layer chromatography, were performed to validate this new method and to compare the relationship between enzymatic activity and substrate degradation. Statistically relevant results were observed for amylase, endoglucanase and polygalacturonase enzymes, even when there was a low substrate concentration in agar, where the halo diameter was high. The results also indicated that the concentrations for efficient enzyme index measurements were 4 g/L carboxymethylcellulose for endoglucanase detection and 8 g/L for amylase and polygalacturonase assays. The results were presented according to the traditional methods for detection of enzymatic activity. This new method can be used as a general test for the detection of important industrial hydrolases. It is a faster and less costly alternative for screening microbial enzyme producing microorganisms and is useful for studying the production of microbial enzymes under different growing conditions.

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.