Enzymatic Treatments for Biosolids: An Outlook and Recent Trends.

Wastewaters are nutrient-rich organic materials containing significant concentrations of different nutrients, dissolved and particulate matter, microorganisms, solids, heavy metals, and organic pollutants, including aromatic xenobiotics. This variety makes wastewater treatment a technological challenge. As a result of wastewater treatment, biosolids are generated. Biosolids, commonly called sewage sludge, result from treating and processing wastewater residuals. Increased biosolids, or activated sludge, from wastewater treatment is a major environmental and social problem. Therefore, sustainable and energy-efficient wastewater treatment systems must address the water crisis and environmental deterioration. Although research on wastewater has received increasing attention worldwide, the significance of biosolids treatments and valorization is still poorly understood in terms of obtaining value-added products. Hence, in this review, we established some leading technologies (physical, chemical, and biological) for biosolids pretreatment. Later, the research focuses on natural treatment by fungal enzymes to end with lignocellulosic materials and xenobiotic compounds (polyaromatic hydrocarbons) as a carbon source to obtain biobased chemicals. Finally, this review discussed some recent trends and promising renewable resources within the biorefinery approach for bio-waste conversion to value-added by-products.

Bioprospecting for fungal enzymes for applications in microalgal biomass biorefineries.

Microalgal biomass is a promising feedstock for biofuels, feed/food, and biomaterials. However, while production and commercialization of single-product commodities are still not economically viable, obtaining multiple products in a biomass biorefinery faces several techno-economic challenges. The aim of this study was to identify a suitable source of hydrolytic enzymes for algal biomass saccharification. Screening of twenty-six fungal isolates for secreted enzymes activity on Chlamydomonas reinhardtii biomass resulted in the identification of Aspergillus niger IB-34 as a candidate strain. Solid-state fermentation on wheat bran produced the most active enzyme preparations. From sixty-five proteins identified by liquid chromatography coupled to mass spectrometry (LC-MS) (ProteomeXchange, identifier PXD034998) from A. niger IB-34, the majority corresponded to predicted secreted proteins belonging to the Gene Ontology categories of catalytic activity/hydrolase activity on glycosyl and O-glycosyl compounds. Skimmed biomass of biotechnologically relevant strains towards the production of commodities, Chlorella sorokiniana and Scenedesmus obliquus, was fully saccharified after a mild pretreatment at 80 °C for 10 min, at a high biomass load of 10% (w/v). The soluble liquid stream, after skimming and saccharification of biomass of both strains, was further converted into ethanol by fermentation with Saccharomyces cerevisiae at a theoretical maximum efficiency, in a separated saccharification and fermentation assays. The resulting insoluble protein, after biomass skimming with an organic solvent and enzymatic saccharification, was highly digestible in an in vitro digestion assay. Proof of concept is presented for an enzyme-assisted biomass biorefinery recovering 81% of the main biomass fractions in a likely suitable form for the conversion of lipids and carbohydrates into biofuels and proteins into feed/food. KEY POINTS: • Twenty-six fungal extracts were analyzed for saccharification of microalgal biomass. • Skimmed biomass was fully enzymatically saccharified and fermented into ethanol. • Up to 81% recovery of biomass fractions suitable for biofuels and feed/food.

Interspecific evolutionary relationships of alpha-glucuronidase in the genus Aspergillus.

The increased availability and production of lignocellulosic agroindustrial wastes has originated proposals for their use as raw material to obtain biofuels (ethanol and biodiesel) or derived products. However, for biomass generated from lignocellulosic residues to be successfully degraded, in most cases it requires a physical (thermal), chemical, or enzymatic pretreatment before the application of microbial or enzymatic fermentation technologies (biocatalysis). In the context of enzymatic technologies, fungi have demonstrated to produce enzymes capable of degrading polysaccharides like cellulose, hemicelluloses and pectin. Because of this ability for degrading lignocellulosic material, researchers are making efforts to isolate and identify fungal enzymes that could have a better activity for the degradation of plant cell walls and agroindustrial biomass. We performed an in silico analysis of alpha-glucoronidase in 82 accessions of the genus Aspergillus. The constructed dendrograms of amino acid sequences defined the formation of 6 groups (I, II, III, IV, V, and VI), which demonstrates the high diversity of the enzyme. Despite this ample divergence between enzyme groups, our 3D structure modeling showed both conservation and differences in amino acid residues participating in enzyme-substrate binding, which indicates the possibility that some enzymes are functionally specialized for the specific degradation of a substrate depending on the genetics of each species in the genus and the condition of the habitat where they evolved. The identification of alpha-glucuronidase isoenzymes would allow future use of genetic engineering and biocatalysis technologies aimed at specific production of the enzyme for its use in biotransformation.

Potencial de los hongos anamorfos de Guatemala para la producción de α-amilasas utilizando como sustrato cascarilla de arroz / Guatemalan anamorphic fungi and their potential use in production of α-amylases using rice husk as a substrate

Los desechos agroindustriales se generan en grandes cantidades, y en la mayoría de los casos son depositados en vertederos lo cual constituye un problema ambiental. Estos residuos lignocelulósicos pueden utilizarse como materia prima o sustrato de crecimiento de hongos anamorfos, que a través de procesos de fermentación pueden producir biocombustibles, enzimas, vitaminas, antioxidantes, alimentos para animales, antibióticos y otros productos químicos. En este estudio se determinó la capacidad de producción de α-amilasas de 20 cepas nativas de hongos anamorfos del cepario de hongos del Departamento de Microbiología, Facultad de Ciencias Químicas y Farmacia, de la USAC a través de fermentación en estado sólido, utilizando como sustrato cascarilla de arroz. La extracción de las enzimas se realizó por microfiltración y la actividad amilolítica fue medida por espectrofotometría. De las cepas evaluadas se encontró que las amilasas de Aspergillus sp. SL15319 mostraron la mayor actividad media (desviación estándar), tanto libres, 930.26 (1.56) UA/dl, como inmovilizadas, 900.34 (3.21) UA/dl, seguido por las de Beltrania rhombica, 905.02 (10.72) y 879.07 (3.87) UA/dl y Aspergillus sp. SL15119, 907.46 (5.17) y 875.95 (9.39) UA/dl (p < .05). La importancia de este estudio radica en dar a conocer el potencial de los hongos anamorfos nativos de Guatemala para el aprovechamiento de los residuos agroindustriales como materia prima para la producción de sustancias de utilidad para el ser humano, y en la reducción de la carga contaminante que se desecha al medio ambiente.

Agroindustrial wastes are generated in large quantities and in most cases deposited in landfills as waste. These lignocellulosic residues can be raw material or substrate for anamorphic fungi, which through fermentation processes can produce biofuels, enzymes, vitamins, antioxidants, animal feed, antibiotics and other chemical products. In this study, the α-amylase production capacity of 20 native strains of anamorphic fungi from the fungal strain collection of Departamento de Microbiología, Facultad de Ciencias Químicas y Farmacia, USAC was determined through solid state fermentation, using rice husk as a substrate. The extraction of the enzymes was carried out by microfiltration and the amylolytic activity was measured by spectrophotometry. Of the strains evaluated, it was found that the amylases of Aspergillus sp. SL15319 showed the highest mean activity (standard deviation), both free, 930.26 (1.56) UA/dl, and immobilized, 900.34 (3.21) UA/dl, followed by those of Beltrania rhombica, 905.02 (10.72) and 879.07 (3.87) UA/dl and Aspergillus sp. SL15119 907.46 (5.17) and 875.95 (9.39) UA/dl (p < .05). The importance of this study lies in making known the potential of native anamorphic fungi in Guatemala for the use of agro-industrial waste as a raw material for the production of substances of use to humans, and in reducing the pollutant load that is discharged into the environment.

Extracellular enzymes of Colletotrichum fructicola isolates associated to Apple bitter rot and Glomerella leaf spot.

Colletotrichum fructicola causes two important diseases on apple in Southern Brazil, bitter rot (ABR) and Glomerella leaf spot (GLS). In this pathosystem, the Colletotrichum ability to cause different symptoms could be related to differences of extracellular enzymes produced by the fungi. Thus, the objectives of this study were to compare the production of these enzymes between ABR- and GLS-isolate in vitro and to evaluate their involvement on infected apple leaves with C. fructicola. In agar plate enzymatic assay, ABR- showed significantly higher amylolytic and pectolytic activity than GLS-isolate. In contrast, for lipolytic and proteolytic no significant differences were observed between isolates. In culture broth, ABR-isolate also had higher activity of pectin lyase (PNL), polygalacturonase (PG) and laccase (LAC). Notably, LAC was significantly five-fold higher in ABR- than GLS-isolate. On the other hand, in infected apple leaves no significant difference was observed between isolates for PNL, PG and LAC. Although differences in extracellular enzymes of ABR- and GLS-isolate have not been observed in vivo, these results contributed to highlight the importance to investigate such enzymes in depth.

Secreted fungal aspartic proteases: A review.

The aspartic proteases, also called aspartyl and aspartate proteases or acid proteases (E.C.3.4.23), belong to the endopeptidase family and are characterized by the conserved sequence Asp-Gly-Thr at the active site. These enzymes are found in a wide variety of microorganisms in which they perform important functions related to nutrition and pathogenesis. In addition, their high activity and stability at acid pH make them attractive for industrial application in the food industry; specifically, they are used as milk-coagulating agents in cheese production or serve to improve the taste of some foods. This review presents an analysis of the characteristics and properties of secreted microbial aspartic proteases and their potential for commercial application.

Screening of Mucor spp. for the production of amylase, lipase, polygalacturonase and protease

Fungi are well known by their ability to excrete enzymes into the environment. Among them, representatives of Mucor Fresen. have important biotechnological potential and some of them produce industrial enzymes. This work studied amylase, lipase, polygalacturonase and protease production by fifty-six isolates of Mucor belonging to 11 different taxa, selected from herbivores dung using solid media. The results showed that the majority of the isolates presented several enzymatic activities with predominance of polygalacturonase (96%), followed by amylase (84%), protease (82%) and lipase (66%).

Os fungos apresentam a capacidade de produzir e secretar enzimas para o meio ambiente. Entre esses, representantes de Mucor Fresen constituem um grupo de microrganismos com importante potencial biotecnológico, sendo responsáveis pela produção de várias enzimas usadas em processos industriais. Foi observado que 56 isolados do gênero Mucor, totalizando 11 táxons, obtidos de fezes de herbívoros são capazes de produzir amilase, lipase, polygalacturonase e protease em meios sólidos. Os resultados demonstraram que 96% dos isolados produziram poligalacturonase, (84%) amilase, (82%) protease e (66%) lipase.