Diverse biological activities and secondary metabolites profile of Penicillium brevicompactum HE19ct isolated from the high-Andean medicinal plant Perezia coerulescens.

Endophytic fungi produced attractive primary and secondary metabolites for industries, pharmacology, and biotechnology. The bioactive potential of HE19ct, identified as Penicillium brevicompactum according to ITS-BenA-caM, was addressed. Antimicrobial and antioxidant activities and secondary metabolite contents using four culture media in Agar-plate (ApF) and Submerged (SmF) fermentation were evaluated. Some plant growth-promoting (PGP) traits and their related genes were tested. HE19ct exhibited antimicrobial activity against Staphylococcus aureus, Enterococcus faecalis, Candida albicans, C. tropicalis, Fusarium sp., Geotrichum candidum, and Alternaria sp. All cultures showed DPPH scavenging activity and phenolic compounds, where ethyl acetate extract of SmF with malt extract showed higher activity and SmF/ApF with potato-dextrose exhibited higher yield, respectively. HE19ct solubilized tricalcium-phosphate and produced siderophore, endoglucanase, proteinase, and amylase. It enhanced the alfalfa's germination at 15 °C, root development, and phenols production at 15 and 24 °C. Phenols, tannins, anthraquinones, triterpenoids/steroids, and alkaloids production were detected depending on culture media. Polyketide synthase type I gene (PksI), subtilisin-like protease prb 1 (Pbr), and siderophore D (sidD) were PCR-amplified. Finally, HE19CT could be a promising source of interesting bioactive compounds for pharmacology and agriculture mainly in extreme conditions, then metabolomic and functional genetic research must be performed to support their appropriate application.

Phytochemical analysis for ten Peruvian Mentheae (Lamiaceae) by liquid chromatography associated with high resolution mass spectrometry.

The profile of secondary metabolites in ten members of tribe Mentheae (Nepetoideae, Lamiaceae) from Peru by liquid chromatography associated with high resolution mass spectrometry, is presented. Salvianolic acids and their precursors were found, particularly rosmarinic acid, caffeic acid ester derivatives, as well as a diversity of free and glycosylated flavonoids as main substances. At all, 111 structures were tentatively identified.

Insight into the genome data of commercially important giant kelp Macrocystis pyrifera.

Kelps or brown algae are a wide group of marine macroalgae that play an important role in aquatic ecosystems and generally have high commercial value. To facilitate brown algal studies, we report the complete genome sequence of the largest kelp Macrocystis pyrifera. The whole genome is ∼428 Mb in size, comprises 44,307 scaffolds with an average GC content of 47%, and is predicted to contain a total of 24,778 genes. 18S sequence-based phylogenetic analysis revealed that littoral brown seaweed Scytosiphon lomentaria is the closest species of M. pyrifera. Numerous genes identified in this dataset are involved in genetic information processing, signaling, and cellular processes, carbohydrate metabolism, and terpenoids biosynthesis.

Comparison of Pigment Production by Filamentous Fungal Strains under Submerged (SmF) and Surface Adhesion Fermentation (SAF).

Although synthetic colorants are widely used in many industries due to their high stability at different conditions in industrial processes, evidence of its negative impact on health and the environment is undeniable. Filamentous fungi are well known for their use as alternative sources to produce natural pigments. However, an adequate comparison of the productivity parameters between the fermentation systems could be limited to their heterogeneous conditions. Even though Solid-State Fermentations (SSF) on natural substrates are widely used for pigments production, complex media, and non-controlled variables (T, pH, medium composition), these systems could not only hamper the finding of accurate productivity parameters, but also mathematical modeling and genomics-based optimization. In this context, the present study screened five pigment-producing fungi by comparing Submerged (SmF) and Surface Adhesion Fermentation [biofilm (BF) and Solid-State (SSF)] with defined media and controlled variables. For this purpose, we used the same defined media with sucrose as the carbon source for pigment production on SmF, BF, and SSF, and BF and SSF were carried out on inert supports. Five molecularly identified Penicillium and Talaromyces strains isolated from the Peruvian rainforest were selected for their ability to produce yellowish-orange colorants. Highest productivities were obtained from T. brunneus LMB-HP43 in SmF (0.18 AU/L/h) and SSF (0.17 AU/L/h), and P. mallochii LMB-HP37 in SSF (0.18 AU/L/h). Both strains also exhibited the highest yields (AU/g biomass) in the three fermentation systems, reaching values greater than 18-folds in SSF compared to the other strains. Conversely, T. wortmannii LMB-HP14 and P. maximae LMB-HP33 showed no ability to produce pigments in the SSF system. The performed experiments accurately compared the effect of the fermentation system on yield and productivity. From this, further genomics approaches can be considered for an extensive analysis of pigment synthesis pathways and a genomics-driven optimization in the best fermentation system.

Phytochemical profile and rosmarinic acid purification from two Peruvian Lepechinia Willd. species (Salviinae, Mentheae, Lamiaceae).

The phytochemical profile of Lepechinia meyenii (Walp.) Epling and Lepechina floribunda (Benth.) Epling obtained by liquid chromatography associated with high-resolution mass spectrometry is presented. Forty eight compounds were detected exhibiting a variety of salvianolic acids and abietane phenolic diterpenoids. A simple procedure by cold evaporative crystallization to purify rosmarinic acid from these botanical species was also shown.

Gene Expression Analysis of Non-Clinical Strain of Aspergillus fumigatus (LMB-35Aa): Does Biofilm Affect Virulence?

Aspergillus fumigatus LMB-35Aa, a saprophytic fungus, was used for cellulase production through biofilms cultures. Since biofilms usually favor virulence in clinical strains, the expression of the related genes of the LMB 35-Aa strain was analyzed by qPCR from the biomass of planktonic cultures and biofilms developed on polyester cloth and polystyrene microplates. For this, virulence-related genes reported for the clinical strain Af293 were searched in A. fumigatus LMB 35-Aa genome, and 15 genes were identified including those for the synthesis of cell wall components, hydrophobins, invasins, efflux transporters, mycotoxins and regulators. When compared with planktonic cultures at 37 °C, invasin gene calA was upregulated in both types of biofilm and efflux transporter genes mdr4 and atrF were predominantly upregulated in biofilms on polystyrene, while aspHs and ftmA were upregulated only in biofilms formed on polyester. Regarding the transcription regulators, laeA was downregulated in biofilms, and medA did not show a significant change. The effect of temperature was also evaluated by comparing the biofilms grown on polyester at 37 vs. 28 °C. Non-significant changes at the expression level were found for most genes evaluated, except for atrF, gliZ and medA, which were significantly downregulated at 37 °C. According to these results, virulence appears to depend on the interaction of several factors in addition to biofilms and growth temperature.

Diversity of endophytic plant-growth microorganisms from Gentianella weberbaueri and Valeriana pycnantha, highland Peruvian medicinal plants.

Microbial diversity in Peruvian mountain areas is poorly know, specially endophytic microorganisms of medicinal native plants from the Cordillera Blanca. So, nine bacterial and six fungal species were isolated from Gentianella weberbaueri and Valeriana pycnantha. According to 16S rDNA analysis, bacterial strains belong to genera Rahnella, Pseudomonas, Serratia, Rouxiella, and Bacillus; while ITS analysis showed that fungi belong to Pyrenochaeta, Scleroconidioma, Cryptococcus, and Plenodomus genera. Rahnella sp. GT24B and P. trivialis VT20B solubilized tricalcium phosphate and produced siderophores at 10 and 24 °C. Five bacteria strains produced indol-3-acetic acid (IAA) at 10 and 24 °C, where Rahnella sp. VT19B showed more production at 10 °C than 24 °C. Rahnella sp. GT24B, Serratia sp. VT28B, and Rahnella sp. GT25B inhibited Fusarium oxysporum growth up to 100, 78 and 74 %, respectively. R. inusitata VT25B and B. licheniformis GT10B showed high cellulolytic and proteolytic activities. On the other hand, only a few fungi moderately inhibited growth of F. oxysporum, and produced siderophores and cellulases. Most of bacteria inoculated on Medicago sativa "alfalfa" and Triticum aestivum "wheat" seeds got better root development, especially Rahnella sp. GT24B, Rouxiella sp.VT24B, Serratia sp. VT28B, and Rahnella sp. VT34B. Finally, this study is the first report of endophytic microorganisms associated to wild medicinal high-mountain Peruvian plants and it show a valuable microbial diversity and its possible role in promoting growth of crops and wild medicinal plants.

Isolation of thermotolerant Bacillus subtilis DCH4 from Chancos hot spring (Carhuaz, Peru) with potential to degrade lignocellulosic agriculture wastes / Aislamiento de Bacillus subtilis DCH4 termotolerante de la fuente termal Chancos (Carhuaz, Perú) con potencial para degradar residuos lignocelulósicos agrícolas

Abstract It was isolated bacteria strains from three different types of samples: fresh water, in situ baits and ex situ enrichment. Serial dilutions were prepared and culture was carried at 50 °C using a Basal-Saline medium. Isolated strains were screened for endoglucanase and xylanase activities with qualitative (Congo Red) and quantitative (DNS) methods. Molecular 16S rDNA sequencing analysis was performed for taxonomic identification. It was isolated 31 strains of which 14 showed hydrolytic activities and belonged to Bacillus subtilis and Bacillus licheniformis species. Moreover, the strain B. subtilis DCH4 showed the highest endoglucanase activity at 45°C and pH 5, and xylanase activity at 55°C and pH 6. Then, DCH4 was cultivated by submerged fermentation with two different media supplemented with sugar cane bagasse, wheat straw, or quinoa stalk to evaluate its saccharification capability. Likewise, it was screening its xylanase and cellulase genes employing specific primers; the amplicons obtained were sequenced, and analyzed. It was found that, enzymatic extracts of DCH4 prepared with cane bagasse or quinoa stalk media achieved the highest endoglucanase and xylanase activities. According to molecular analysis of genes involved in the hydrolytic process, the endoglucanase and xylanase activities exhibited by DCH4 could be attributed to a bifunctional cellulase conformed by endo-beta-1,4-glucanase (GH5) joined to cellulose binding domain 3 (CBM3), and an endo-1,4-beta-xylanase (GH11), respectively. Further transcriptomic experiments would be considered to accomplish optimization strategies for biofuel production from lignocellulosic biomass.

Resumen Se aislaron cepas de bacterias provenientes de tres tipos de muestras: agua fresca, cebos enriquecidos in situ y ex situ. Se prepararon diluciones seriadas y el cultivo fue a 50 °C usando un medio Salino-Basal. Las cepas aisladas fueron tamizadas para las actividades endoglucanasa y xilanasa con métodos cualitativos (Rojo Congo) y cuantitativos (DNS). Se usó el análisis molecular 16S rDNA para la identificación taxonómica. Se aislaron 31 cepas, de las cuales 14 mostraron actividades hidrolíticas y pertenecían a Bacillus subtilis y Bacillus licheniformis. Además, B. subtilis DCH4 mostró la mayor actividad endoglucanasa a 45 °C y pH 5, y xilanasa a 55 °C y pH 6. Entonces, DCH4 se cultivó por fermentación sumergida con dos medios diferentes suplementado con bagazo de caña de azúcar, paja de trigo o tallo de quinua para evaluar su capacidad de sacarificación. También, se exploraron los genes de xilanasa y celulasa mediante cebadores específicos; los amplicones obtenidos fueron secuenciados y analizados. Se encontró que los extractos enzimáticos de DCH4 preparados con bagazo de caña o tallos de quinua mostraron las actividades endoglucanasa y xilanasa más elevadas. De acuerdo a los análisis moleculares de los genes involucrados en el proceso hidrolítico, las actividades de endoglunacasa y xilanasa exhibidas por DCH4 podrían atribuirse a una celulasa bifuncional conformada por una endo-beta-1,4-glucanasa (GH5) unida al dominio celulosa 3 (CBM3), y una endo-1,4-beta-xilanasa (GH11), respectivamente. Posteriores experimentos transcriptómicos podrían ser considerados para lograr estrategias de optimización para la producción de biocombustibles a partir de biomasa lignocelulósica.

A simple and accurate method for specific quantification of biomass in mixed cultures of filamentous fungi by quantitative PCR / Un método simple y preciso para la cuantificación específica de biomasa en cultivos mixtos de hongos filamentosos por PCR cuantitativa

Abstract Production of lignocellulolytic enzymes by filamentous fungi have a great potential at industrial level due to their widespread applications. Mixed fungal cultures and particularly mixed fungal biofilms constitute a promising fermentation system for an enhanced enzyme production. However, it has not been addressed how much of this enhancement depends on the mixed biomass proportion. In this sense, the aim of this study was to develop a method to specifically and accurately quantify mixed fungal biomass. For this purpose, mixed biofilm cultures composed of Aspergillus niger and Trichoderma reesei, two filamentous fungi used industrially for cellulase production, were collected from 48 to 120 h of growth; mycelia were pulverized, and DNA was extracted for qPCR assays with specific primers for each fungus. Primers were designed from non-conserved regions of sequences of actin and β-tubulin genes of both A. niger and T. reesei. Specificity of these primers was tested in silico and experimentally. A statistically significant correlation was obtained between qPCR-calculated biomass and dry weight biomass data. By this method, it was possible to detect changes on mycelia proportions in biofilms over time, suggesting a competitive interaction between these two fungi. In conclusion, this method allows a specific and accurate quantification of mixed fungal biomass and could be also applied to different mixed culture systems for studying microbial interactions.

Resumen La producción de enzimas lignocelulolíticas por hongos filamentosos tiene un gran potencial a nivel industrial debido a sus diversas aplicaciones. Los cultivos fúngicos mixtos y particularmente las biopelículas fúngicas mixtas constituyen un sistema de fermentación prometedor para una mayor producción enzimática. Sin embargo, no se ha abordado cuánto de esta mejora depende de la proporción de biomasa mixta. En este sentido, el objetivo de este estudio fue desarrollar un método para cuantificar de forma específica y precisa la biomasa fúngica mixta. Para este propósito, se recolectaron cultivos mixtos de biopelículas de 48 a 120 h de crecimiento compuestos por Aspergillus niger y Trichoderma reesei, dos hongos filamentosos utilizados industrialmente para la producción de celulasas; el micelio se pulverizó y el ADN se extrajo para ensayos de qPCR con cebadores específicos para cada hongo. Los cebadores se diseñaron a partir de regiones no conservadas de las secuencias de los genes de actina y β-tubulina de A. niger y T. reesei. La especificidad de estos cebadores se probó in silico y experimentalmente. Se obtuvo una correlación estadísticamente significativa entre la biomasa calculada mediante qPCR y los datos de biomasa en peso seco. Mediante este método, fue posible detectar cambios en las proporciones de los micelios en las biopelículas a lo largo del tiempo, lo que sugiere una interacción competitiva entre estos dos hongos. En conclusión, este método permite una cuantificación específica y precisa de la biomasa fúngica mixta y también podría aplicarse a diferentes sistemas de cultivo mixto para estudiar interacciones microbianas.

First global transcriptome analysis of brown algae Macrocystis integrifolia (Phaeophyceae) under marine intertidal conditions.

To understand the physiological responses of the brown macroalga Macrocystis integrifolia during the marine tidal cycle, two RNA libraries were prepared from algal frond samples collected in the intertidal zone (0 m depth) and subtidal zone (10 m depth). Samples collected from intertidal zone during low tide was considered as abiotic stressed (MI0), while samples collected from subtidal zone was considered as control (MI10). Both RNA libraries were sequenced on Illumina NextSeq 500 which generated approx. 46.9 million and 47.7 million raw paired-end reads for MI0 and MI10, respectively. Among the representative transcripts (RTs), a total of 16,398 RTs (39.20%) from MI0 and 21,646 RTs (39.24%) from MI10 were successfully annotated. A total of 535 unigenes (271 upregulated and 264 downregulated) showed significantly altered expression between MI0 and MI10. In abiotic-stressed condition (MI0), the relative expression levels of genes associated with antioxidant defenses (vanadium-dependent bromoperoxidase, glutathione S-transferase, lipoxygenase, serine/threonine-protein kinase, aspartate Aminotransferase, HSPs), water transport (aquaporin), photosynthesis (light-harvesting complex) protein were significantly upregulated, while in control condition (MI10) most of the genes predominantly involved in energy metabolism (NADH-ubiquinone oxidoreductase/NADH dehydrogenase, NAD(P)H-Nitrate reductase, long-chain acyl-CoA synthetase, udp-n-acetylglucosamine pyrophosphorylase) were overexpressed.

High-quality draft genome sequence of a biofilm forming lignocellulolytic Aspergillus niger strain ATCC 10864.

Filamentous fungus Aspergillus niger has high industrial value due to their lignocellulolytic enzyme activities and ATCC 10864 is one of the few type strains of A. niger which has a unique biofilm forming capability. Here we report the first draft genome sequence of A. niger ATCC 10864 strain. The genome of A. niger ATCC 10864 is 36,172,237 bp long and comprise of 310 scaffolds with 49.5% average GC content. A total of 10,804 protein-coding genes were predicted among which 10,761 genes were with putative functions. A. niger ATCC 10864 genome coded for 709 putative carbohydrate active enzyme families distributed in six functional categories and among them glycoside hydrolases (GHs) represent the most number of families (279). Genes that include pepA, brlA, exgA, LaeA, rodA, GCN have also been identified in this study, which may play a role in biofilm formation. This high-quality draft genome sequence will facilitate our understanding of the mechanisms behind fungal biofilm formation and higher lignocellulolytic enzyme production.

Insights from the genome of a high alkaline cellulase producing Aspergillus fumigatus strain obtained from Peruvian Amazon rainforest.

Here, we report the complete genome sequence of a high alkaline cellulase producing Aspergillus fumigatus strain LMB-35Aa isolated from soil of Peruvian Amazon rainforest. The genome is ∼27.5mb in size, comprises of 228 scaffolds with an average GC content of 50%, and is predicted to contain a total of 8660 protein-coding genes. Of which, 6156 are with known function; it codes for 607 putative CAZymes families potentially involved in carbohydrate metabolism. Several important cellulose degrading genes, such as endoglucanase A, endoglucanase B, endoglucanase D and beta-glucosidase, are also identified. The genome of A. fumigatus strain LMB-35Aa represents the first whole sequenced genome of non-clinical, high cellulase producing A. fumigatus strain isolated from forest soil.

Metagenomic analysis of microbial community of an Amazonian geothermal spring in Peru.

Aguas Calientes (AC) is an isolated geothermal spring located deep into the Amazon rainforest (7°21'12″ S, 75°00'54″ W) of Peru. This geothermal spring is slightly acidic (pH 5.0-7.0) in nature, with temperatures varying from 45 to 90 °C and continually fed by plant litter, resulting in a relatively high degree of total organic content (TOC). Pooled water sample was analyzed at 16S rRNA V3-V4 hypervariable region by amplicon metagenome sequencing on Illumina HiSeq platform. A total of 2,976,534 paired ends reads were generated which were assigned into 5434 numbers of OTUs. All the resulting 16S rRNA fragments were then classified into 58 bacterial phyla and 2 archaeal phyla. Proteobacteria (88.06%) was found to be the highest represented phyla followed by Thermi (6.43%), Firmicutes (3.41%) and Aquificae (1.10%), respectively. Crenarchaeota and Euryarchaeota were the only 2 archaeal phyla detected in this study with low abundance. Metagenomic sequences were deposited to SRA database which is available at NCBI with accession number SRX1809286. Functional categorization of the assigned OTUs was performed using PICRUSt tool. In COG analysis "Amino acid transport and metabolism" (8.5%) was found to be the highest represented category whereas among predicted KEGG pathways "Metabolism" (50.6%) was the most abundant. This is the first report of a high resolution microbial phylogenetic profile of an Amazonian hot spring.

Production of alkaline cellulase by fungi isolated from an undisturbed rain forest of peru.

Alkaline cellulase producing fungi were isolated from soils of an undisturbed rain forest of Peru. The soil dilution plate method was used for the enumeration and isolation of fast growing cellulolytic fungi on an enriched selective medium. Eleven out of 50 different morphological colonies were finally selected by using the plate clearing assay with CMC as substrate at different pH values. All 11 strains produced cellulases in liquid culture with activities at alkaline pH values without an apparent decrease of them indicating that they are true alkaline cellulase producers. Aspergillus sp. LM-HP32, Penicillium sp. LM-HP33, and Penicillium sp. LM-HP37 were the best producers of FP cellulase (>3 U mL(-1)) with higher specific productivities (>30 U g(-1) h(-1)). Three strains have been found suitable for developing processes for alkaline cellulase production. Soils from Amazonian rain forests are good sources of industrial fungi with particular characteristics. The results of the present study are of commercial and biological interest. Alkaline cellulases may be used in the polishing and washing of denim processing of the textile industry.

Recent advances on filamentous fungal biofilms for industrial uses.

Industrial enzymes are produced by submerged fermentation (SF) and by solid-state fermentation (SSF) to a lesser extent. Although SSF has several advantages, its scale-up is difficult. The role of physiological and genetic properties of microorganisms growing attached to surfaces could explain the advantages of SSF. Filamentous fungi are naturally adapted to growth on surfaces and in these conditions they show a particular physiological behavior which is different from that in SF; thus, they also form biofilms. Fermentation by filamentous fungal biofilms (FFB) is a homogeneous production system within a liquid environment based on the infrastructure of the SF process with the productive efficiency of the SSF. Enzyme production levels of FFB are much higher than those obtained in SF and they are also amenable of mixed fungal cultivation. Transcriptomic and proteomic tools are used to uncover the fundamental biological issues behind FFB. Several genes encoding cellulolytic enzymes are either differentially expressed or overexpressed in FFB. Moreover, our proteomic studies of Aspergillus niger biofilms compared to SF indicate that many intracellular proteins are either differentially expressed or overexpressed. Clinically important fungi like A. fumigatus also form biofilms when they infect lungs and recent studies demonstrate same gene expression features. These results support our hypothesis of cell adhesion and its role in the new schemes for improved fermentative production of industrial enzymes.

Efecto de diámetro de esfera y densidad celular en la producción de etanol con levadura inmovilizada en alginato / Effect of pellet diameter and cell density on ethanol production with alginate immobilized yeast

Las células inmovilizadas tienen aplicación potencial en la producción de biocombustibles posibilitando la reutilización de biomasa, el empleo de diversas configuraciones de reactores y sistemas de cultivo, el manejo de altas densidades celulares alcanzando altas productividades volumétricas, y la simplificación de operaciones de procesamiento de salida. El objetivo del presente estudio fue evaluar la influencia del diámetro de las perlas y la densidad celular en la producción de etanol con Saccharomyces uvarum inmovilizada en alginato al 2% (p/v). Para ello se evaluaron tres diámetros de perlas de 2, 2,5 y 3 mm. Las células inmovilizadas fueron cultivadas en medio con 12% (p/v) de glucosa en biorreactores de columna sin agitación a 28 ºC, y se operaron cuatro lotes consecutivos de 48 horas cada uno. En cada lote se cuantificó el consumo de glucosa y se determinó la cantidad de etanol producido. Los rendimientos máximos de etanol para las esferas de 2, 2,5 y 3 mm de diámetro fueron 81, 83 y 97% del rendimiento teórico. La máxima productividad volumétrica de etanol fue 1,2 g/L-1/h-1 con un consumo de glucosa de 99,8% al término del lote, correspondiente a las columnas con perlas de 3 mm y con una producción de 0,017 g de etanol por esfera. La producción de etanol acumulada en cada sistema fue 178, 189 y 200 g/L-1 para 2, 2,5 y 3 mm respectivamente, encontrándose una relación directa con el diámetro de perla e inversa respecto a la densidad celular. Los rendimientos de etanol obtenidos son superiores a los reportados para la misma especie.

Immobilized cells have a potential use in biofuel production. They also allow re-using biomass, using diverse reactor configurations and culture systems, handling high cell densities to obtain high volumetric productivities and to simplify the downstream processing. The purpose of this work was to evaluate the influence of bead diameter and cell density on ethanol production using immobilized Saccharomyces uvarum in 2% (w/v) alginate. For that, three bead diameters (2, 2.5 and 3 mm) were evaluated. Immobilized cells were cultured on a 12% (w/v) glucose medium in column bioreactors without agitation at 28 °C for four 48 h–repeated batches. For each batch, both glucose consumption and ethanol produced were measured. Maximum yields for 2, 2.5 and 3 mm bead diameters were 81, 83 and 97% of theoretical yield. Maximum volumetric productivity of ethanol was 1.2 g/L-1/h-1 with 99.8% glucose consumption at the end of the batch, corresponding to the 3 mm bead diameter and the ethanol production per bead was 0.017 g. Accumulated ethanol production for each system was 178, 189 and 200 g/L-1 for 2, 2.5 y 3 mm bead diameter, respectively, being this directly related to bead diameter and inversely related to cell density. Ethanol yields were higher than those reported for the same species.

Influencia de la concentración de inóculo en la producción de celulasa y xilanasa por aspergillus niger / Influence of inoculum concentration on the cellulase and xylanase production by aspergillus niger

Existe un gran interés por el uso de enzimas lignocelulolíticas en varias industrias, y en la biodegradación de biomasa para la producción de biocombustibles y otras aplicaciones. Entre las fuentes microbianas de enzimas, Aspergillus niger es uno de los microorganismos más utilizados en la producción de enzimas industriales, debido a sus niveles altos de secreción de proteína y a su condición GRAS (generally regarded as safe). El objetivo del presente estudio fue evaluar la influencia de la concentración de inóculo en la morfología y producción de celulasas y xilanasas con A. niger en cultivo sumergido. Para ello, fueron inoculados matraces de 250 mL con 40 mL de medio con 3% (v/v) de una suspensión de 104 o 108 esporas por mililitro e incubados a 28 ºC y 175 rpm durante 120 horas. Se utilizaron 10 g*L-1 de lactosa como fuente de carbono. En cada caso se determinó la cantidad de biomasa, la proteína extracelular soluble, lactosa residual, actividad celulasa total y xilanasa cada 24 horas. Aunque no hubo un efecto notorio en la morfología de crecimiento, salvo en el color y el diámetro de pellets obtenidos, sí se afectó la µmax (0,06 y 0,03 h-1 para 104 y 108 esporas*mL-1, respectivamente) y la concentración máxima de biomasa. Además, mientras que las productividades volumétricas de celulasa (ΓFPA) (8,2 y 8,0 UI.*L-1*h-1 para 104 y 108 esporas*mL-1, respectivamente) fueron similares para ambos inóculos, la productividad de xilanasa (ΓXIL) fue mayor para el inóculo más concentrado (29,7 y 33,4 UI¨*L-1*h-1 para 104 y 108 esporas*mL-1, respectivamente). Los resultados indican que la productividad de celulasas y xilanasas está estrechamente relacionada con la concentración de inóculo.

There is a great interest for the use of lignocellulolytic enzymes in several industries and in biomass degradation for production of biofuels and other applications. Among the microbial sources of enzymes, Aspergillus niger is one of the most used microorganisms in the production of industrial enzymes due to its high levels of protein secretion and its GRAS (generally regarded as safe) condition. The aim of the present study was to evaluate the influence of A. niger inoculum concentration in the morphology and production of cellulases and xylanases in submerged cultures. For this, 250 mL flasks containing 40 mL culture medium were inoculated with a 3% (v/v) of either 104 or 108 spores per milliliter suspension and incubated at 28 º C and 175 rpm during 120 hours. Lactose (10 g*L-1) was used as the carbon source. In each case, the amount of biomass, the extracellular soluble protein, residual lactose, total celullase activity and xylanase activity were determined every 24 hours. Even thought there was not a notorious effect on the growth morphology, except in color and diameter of pellets; µmax was affected (0.06 and 0.03 h-1 for 104 and 108 spores*mL-1, respectively) as well as maximum biomass concentration. In addition, while the volumetric productivity of cellulase (8.2 and 8.0 UI*L-1*h-1 for 104 and 108 spores*mL-1, respectively) were similar for both inocula, the productivity of xylanase was greater for the more concentrated inoculum (29.7 and 33.4 UI*L-1*h-1 for 104 and 108 spores*mL-1, respectively).The results show that cellulase and xylanase productivities are closely related to the inoculum concentration.

Cellulase production by Aspergillus niger in biofilm, solid-state, and submerged fermentations.

Cellulase production by Aspergillus niger was compared in three different culture systems: biofilm, solid-state, and submerged fermentation. Biofilm and solid-state fermentations were carried out on perlite as inert support, and lactose was used as a carbon source in the three culture systems. In cryo-scanning electron microscopy, biofilm and solid-state cultures gave similar morphological patterns and confirmed that both spore first attachment and hyphal adhered growth are helped by the production of an adhesive extracellular matrix. Biofilm cultures produced higher cellulase activities than those in submerged and solid-state cultures (1,768, 1,165, and 1,174 U l(-1), respectively). Although biofilm cultures grew less than the other cultures, they produced significantly higher cellulase yields (370, 212, and 217 U g(-1) lactose, respectively) and volumetric productivities (24, 16, and 16 U l(-1) h(-1), respectively). Likewise, endoglucanase and xylanase activities were higher in biofilm cultures. Under the conditions tested, it seems that fungal attached growth on perlite may favor better enzyme production. Biofilms are efficient systems for cellulase production and may replace solid-state fermentation. Biofilm fermentation holds promise for further optimization and development. The results of this work reveal that fungal biofilms may be used for the commercial production of cellulase employing the technology developed for submerged fermentation at high cell densities.

Production of lignocellulolytic enzymes by Aspergillus niger biofilms at variable water activities

Lignocellulolytic enzyme production by Aspergillus niger was compared both in submerged fermentation (SF) and biofilm fermentation (BF) at varying water activities. Maximal filter paper activity, endoglucanase and xylanase activities were much higher in BF (2.96, 4.7 and 4.61 IU ml-1, respectively) than in SF cultures (1.71, 1.31 and 2.3 IU ml-1, respectively) but biomass yields were lower in BF than in SF (0.338 g g-1 and 0.431 g g-1, respectively). In the presence of 20 percent ethylene glycol (a w = 0.942) the enzyme activities decreased in both systems but BF still had higher levels (1.0, 1.0 and 2.6 IU ml-1, respectively) than SF cultures (0.6, 0.7 and 1.5 IU ml-1, respectively). An increase in xylanase specific activity of more than 2 fold (from 4.2 to 10.2 IU mg-1 biomass) was observed in the presence of 20 percent ethylene glycol, suggesting differential regulatory mechanisms in biofilm fermentation related to cell adhesion.