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.

Microorganismos tolerantes a metales pesados del pasivo minero Santa Rosa, Jangas (Perú) / Heavy metals tolerant microorganisms from mine tailing wastelands Santa Rosa, Jangas (Peru)

En el presente trabajo estudiamos el grado de tolerancia a metales pesados de hongos y bacterias aisladas de suelos con y sin rizósfera, con el propósito de conocer su potencial para aplicaciones en biorremediación. Las muestras procedían del pasivo minero de Santa Rosa de Jangas. Los hongos y bacterias aislados fueron identificados taxonómicamente mediante el análisis de la región ITS y 16S ADNr, respectivamente. El índice de tolerancia a metales pesados se calculó usando medio salino suplementado con 1 mM a plomo (II), cobre (II), niquel (II) o zinc (II), y 0.1 mM a plata (I), cromo (VI) o cadmio (II). En total se aislaron 23 hongos y 18 bacterias. Las cepas de hongos con mejores índices de tolerancia fueron: Fusarium temperatum CTLM05 (Pb+2), Fusarium temperatum CTLM08 (Zn+2), Fusarium oxysporum CTLM18 (Ni+2 y Cd+2), Fusarium oxysporum CTLM12 (Ag+1), Fusarium inflexum CTLM22 (Cu+2) y Penicillium vanluykii CTLM11 (Cr+6). Las cepas de bacterias con mayores índices de tolerancia fueron Bacillus licheniformis SSR18 (Cd+2, Ni+2 y Zn+2), Bacillus subtilis SSR3 (Pb+2), Serratia sp. SSR15 (Cu+2), Serratia sp. SSR13 (Ag+1) y Bacillus cereus SSR01 (Cr+6). También se encontró que los hongos mostraron mejores índices de tolerancia que las bacterias. Finalmente, los suelos del pasivo ambiental minero de Santa Rosa de Jangas poseen una microflora interesante, probablemente con mecanismos para su adaptación, crecimiento y desarrollo sobre metales pesados y pueden ser de utilidad para el desarrollo de procesos biotecnológicos y biorremediación.

In this work, we studied the degree of tolerance to heavy metals of fungi and bacteria isolated from soils with and without rhizosphere, in order to know its potential for applications in bioremediation. The samples came from Santa Rosa de Jangas mining liability. The fungi and bacterial strains were taxonomically identified by ITS region and 16S rDNA analysis, respectively. Heavy metal tolerance indices were calculate using salt medium supplemented with 1mM of lead (II), cupper (II), nickel (II) or zinc (II); and 0.1 mM of silver (I), chromium(VI) or cadmium (II). It was isolated 23 fungi and 18 bacteria strains. The fungi with better tolerance indices were Fusarium temperatum CTLM05 (Pb+2), Fusarium temperatum CTLM08 (Zn+2), Fusarium oxysporum CTLM18 (Ni+2 and Cd+2), Fusarium oxysporum CTLM12 (Ag+1), Fusarium inflexum CTLM22 (Cu+2), and Penicillium vanluykii CTLM11 (Cr+6). Likewise, the bacterial strains with better tolerance indices were Bacillus licheniformis SSR18 (Cd+2, Ni+2 and Zn+2), Bacillus subtilis SSR3 (Pb+2), Serratia sp. SSR15 (Cu+2), Serratia sp. SSR13 (Ag+1) and Bacillus cereus SSR01 (Cr+6). Too, it was found that fungi showed better tolerance indices than bacterial strains. Finally, the soil from Santa Rosa waste mine have an interesting microflora, probably with mechanisms for their adaptation, growth, and development were heavy metals are present and they could be useful to perform biotechnology and bioremediation processes.

Antimicrobial, antioxidant and phytochemical assessment of wild medicinal plants from Cordillera Blanca (Ancash, Peru) / Evaluación antimicrobiana, antioxidante y fitoquímico de plantas medicinales silvestres de la Cordillera Blanca (Ancash, Perú)

Twenty-eight native plants mainly used to cure diseases related to microbial infection and stress oxidative disorders were selected to test the antimicrobial activity against E. coli, P. aeruginosa, S. aureus, B. subtilis, and C. albicans using diffusion and microdilution methods. The antioxidant activity was determined by scavenging DPPH free-radical and phytochemical evaluation was performed for plants with promising activities. Twenty-seven plants showed antibacterial activity, four had anti-Candida activity, and four showed antioxidant activity. It was found that Oreocallis grandiflora, Gentianella weberbaueri, Gamochaeta americana, Hypericum laricifolium, Loricaria ferruginea, Muehlenbeckia volcanica, and Oenothera multicaulis, showed promising biological activity and contained alkaloids, phenolic compounds, flavonoids, catecholic or gallic tannins. This study leaves evidence about the medicinal potential of wild high-Andean plants; thus, further pharmacological, phytochemical, ecological and biotechnological studies will contribute to promote their conservation and sustainable use; especially since they are highly vulnerable and risk extinction.

Se seleccionó veintiocho plantas nativas usadas principalmente para tratarcurar enfermedades relacionadas principalmente con infecciones microbianas y desordenes oxidativos. A estas plantas se para ser evaluóados en su actividad antimicrobiana sobre E. coli, P. auriginosa, S. aureus, B. subtilis, y C. albicans usando métodos de difusión y microdilución. Se determinó la actividad antioxidante mediante el ensayo del libre radical DPPH y se realizó la evaluación fitoquímica de las plantas con actividades promisorias. Veinte siete plantas mostraron actividad antibacteriana, cuatro mostraron actividad anti-Candida, y cuatro actividad antioxidante. Oreocallis grandiflora, Gentianella weberbaueri, Gamochaeta americana, Hypericum laricifolium, Loricaria ferruginea, Muehlenbeckia volcanica, y Oenothera multicaulis mostraron actividad biológica promisoria, y se encontró que contienen alcaloides, compuestos fenólicos, flavonoides, taninos gálicos y catecólicos. Este estudio deja evidencia del potencial medicinal de las plantas silvestres alto andinas; por lo tanto, los estudios farmacológicos, fitoquímicos, ecológicos y biotecnológicos contribuirían en la promoción de su conservación y uso sustentable debido a su alta vulnerabilidad y riesgo de extinción.

Alkaline Cellulase Production by Penicillium mallochii LMB-HP37 Isolated from Soils of a Peruvian Rainforest.

Alkaline cellulases are demanded by the textile industry for several purposes but commercial preparations showing activity at alkaline conditions are very scarce. Aim: To characterize a Penicillium strain isolated form soils of a Peruvian rainforest showing alkaline cellulase activity that may be useful for the textile industry. Methodology: The molecular identification was based on the DNA sequence of its ITS region using ITS1 and ITS4 primers after PCR amplification. Cellulase production was evaluated in shaken flasks by using either lactose or microcrystalline cellulose. Total cellulase (as FPA) and endoglucanase activities were evaluated by the standard methods at several pH levels. Also, the cellulase activity of culture filtrates was tested for antipilling activity as compared to a commercial neutral cellulase preparation. Results: After raw data of ITS DNA sequence was processed, multiple alignment and phylogenetic analysis confirmed that our strain can be named as Penicillium mallochii LMB-HP37. Higher activity was attained for neutral total cellulase on lactose (3371±108 U/l at pH 7.4) and alkaline cellulases attained similar activity levels than the acid cellulase (2978±151 U/l at pH 8.4 and 2910±42 U/l at pH 9.4). FPA and endoglucanase activities were produced at high volumetric (46.8±1.5 and 13.5±1.0 U/l.h, respectively) and specific (32.9±1.1 and 9.5±0.7 U/gbiomass.h, respectively) productivities at the same pHs which indicate that this strain may be suitable for commercial development. The enzyme of P. mallochii LMB-HP37 had slightly better results than the commercial enzyme as an anti-pilling agent even though is a crude preparation. Conclusion: Penicillium mallochii LMB-HP37 produced high total cellulase activity on lactose which compares to well-known cellulase producers but at neutral to alkaline pH levels. Data obtained reveal that the crude enzyme is suitable for anti-pilling process (biopolishing) and may be also useful for biostoning.