RESUMEN
Endophytic bacteria found in marine macroalgae have been studied for their potential antimicrobial activity, consequently, they could serve as a valuable source of bioactive compounds to control pathogenic bacteria, yeasts, and fungi. Algae endophytic bacteria were isolated from Caulerpa sp., Ulva sp., Ahnfeltiopsis sp., and Chondracantus chamissoi from Yacila and Cangrejo Beaches (Piura, Peru). Antimicrobial assays against pathogenic bacteria were evaluated using cross-culture, over-plate, and volatile organic compound tests. Afterward, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of selected crude extracts were determined, also ITS molecular analysis, antifungal activity, and PCR of iturin, fengycin, and surfactin genes were performed for bacteria strains exhibiting better activity. Forty-six algae endophytic bacteria were isolated from algae. Ten strains inhibited gram-positive pathogenic bacteria (Enterococcus faecalis, Staphylococcus epidermidis, S. aureus, and Listeria monocytogenes), and 12 inhibited gram-negative bacteria (Escherichia coli and Salmonella enteric sv typhimurium). Bacteria with better activity belong to Bacillus sp., Kluyvera ascorbata, Pantoea agglomerans, Leclercia adecarboxylata, and Enterobacter sp., which only four showed antifungal activities against Candida albicans, C. tropicalis, Colletotrichium sp., Fusarium sp., Fusarium oxysporum, and Alternaria sp. Furthermore, K. ascorbata YAFE21 and Bacillus sp. YCFE4 exhibited iturin and fengycin genes. The results indicate that the algae endophytic bacteria found in this study, particularly K. ascorbata YAFE21, Bacillus sp. YCFR6, L. adecarboxylata CUFE2, Bacillus sp. YUFE8, Enterobacter sp. YAFL1, and P. agglomerans YAFL6, could be investigated as potential producers of antimicrobial compounds due to their broad activity against various microorganisms.
Asunto(s)
Endófitos , Pruebas de Sensibilidad Microbiana , Algas Marinas , Endófitos/aislamiento & purificación , Endófitos/genética , Endófitos/metabolismo , Endófitos/química , Endófitos/clasificación , Algas Marinas/microbiología , Bacterias/efectos de los fármacos , Bacterias/aislamiento & purificación , Bacterias/clasificación , Antiinfecciosos/farmacología , Antiinfecciosos/aislamiento & purificación , Antibacterianos/farmacología , Antibacterianos/aislamiento & purificación , Antifúngicos/farmacología , Antifúngicos/aislamiento & purificación , Hongos/efectos de los fármacos , Hongos/aislamiento & purificación , Hongos/clasificación , Bacterias Gramnegativas/efectos de los fármacos , Ulva/microbiología , Caulerpa/microbiología , Bacterias Grampositivas/efectos de los fármacosRESUMEN
The lignocellulose bioconversion process is an eco-friendly and green-economy alternative technology that allows the reduction of pollution and global warming, so it is necessary for thermophilic and thermostable hydrolytic enzymes from natural sources. This research aimed to isolate cellulolytic and xylanolytic microbial consortia from Huancarhuaz hot spring (Peru) from sludge or in situ baiting cultured with or without sugarcane bagasse. According to the hydrolytic activities consortium T4 from in situ baiting was selected. It was cultivated in submerged fermentation at 65 °C, pH 6.5 for eight days using LB supplemented with sugar cane bagasse (SCB), pine wood sawdust (PWS), CMC, xylan of birchwood, or micro granular cellulose. Crude extract of culture supplemented with SCB (T4B) showed better endoglucanase and xylanase activities with higher activities at 75 °C and pH 6. In these conditions, cellulase activity was kept up to 57% after 1 h of incubation, while xylanase activity was up to 63% after 72 h. Furthermore, this crude extract released reduced sugars from pretreated SCB and PWS. According to metagenomic analysis of 16S rDNA, Geobacillus was the predominant genus. It was found thermostable genes: a type of endoglucanase (GH5), an endo-xylanase (GH10), and alkali xylanase (GH10) previously reported in Geobacillus sp. strains. Finally, Huancarhuaz hot spring harbors a genetic microbial diversity for lignocellulosic waste bioconversion in high temperatures, and the T4B consortium will be a promising source of novel extreme condition stable enzymes for the saccharification process.
RESUMEN
Endophytic and rhizospheric microorganisms associated with six native plants adapted to heavy metal polluted soil from Punta Olímpica and Chahuapampa, located in Callejón de Huaylas mountains, were evaluated as potential candidates for technologies to clean polluted ecosystems. It was selected 14 bacteria and 9 fungi strains by their iron and/or aluminum siderophore production trait, where BEP17-Dm showed higher production. According to the 16S rDNA analysis, bacteria belong to Pseudomonas, Bacillus, and Achromobacter genera, whereas by ITS analysis fungi belong to Talaromyces, Hypoxylon, Tolypocladium, and Penicillium. All bacteria strains tolerated lead (2-8 mM) and eigth tolerated cadmium (1-6 mM); also all fungi tolerated lead (9-70 mM) and cadmium (3-10 mM). Two bacteria and six fungi solubilized cadmium carbonate, while eleven bacteria and two fungi solubilized tricalcium phosphate, where P. japonica BEP18-Dm and B. subtilis BRU16-Sr exhibited higher solubilization index. None strains solubilized lead carbonate. BEP18-Dm produced higher concentration of IAA (53.42 µgml-1); while six bacteria and all fungi strains produced a low concentration of auxins. Medicago sativa seedlings inoculated with BEP17-Dm, BEP18-Dm, or BRU16-Sr showed more surviving percentage under in vitro culture in presence of Cd, Pb (0.5-1.0 mM), or Al (2.5-5.0 mM). Finally, it is the first report of siderophore-producing microorganisms from polluted soil of Callejón de Huaylas highlands, interestedly they displayed metabolic properties useful to enhance phytoremediation and biotechnology application.
Asunto(s)
Bacterias/metabolismo , Hongos/metabolismo , Metales Pesados/metabolismo , Plantas/microbiología , Sideróforos/genética , Sideróforos/metabolismo , Contaminantes del Suelo , Bacterias/clasificación , Bacterias/genética , Bacterias/aislamiento & purificación , Biodegradación Ambiental , Restauración y Remediación Ambiental , Hongos/clasificación , Hongos/genética , Hongos/aislamiento & purificación , Metales Pesados/análisis , Perú , Raíces de Plantas/microbiología , Microbiología del SueloRESUMEN
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.
Asunto(s)
Bacterias/clasificación , Endófitos/clasificación , Hongos/clasificación , Gentianella/microbiología , Valeriana/microbiología , Bacterias/aislamiento & purificación , Productos Agrícolas/crecimiento & desarrollo , Endófitos/aislamiento & purificación , Hongos/aislamiento & purificación , Gentianella/crecimiento & desarrollo , Ácidos Indolacéticos/metabolismo , Perú , Filogenia , Raíces de Plantas/microbiología , Plantas Medicinales/crecimiento & desarrollo , Plantas Medicinales/microbiología , ARN Ribosómico 16S/genética , Valeriana/crecimiento & desarrolloRESUMEN
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.
RESUMEN
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.
RESUMEN
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 seleccionoÌ veintiocho plantas nativas usadas principalmente para tratarcurar enfermedades relacionadas principalmente con infecciones microbianas y desordenes oxidativos. A estas plantas se para ser evaluoÌados en su actividad antimicrobiana sobre E. coli, P. auriginosa, S. aureus, B. subtilis, y C. albicans usando meÌtodos de difusioÌn y microdilucioÌn. Se determinoÌ la actividad antioxidante mediante el ensayo del libre radical DPPH y se realizoÌ la evaluacioÌn fitoquiÌmica 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 bioloÌgica promisoria, y se encontroÌ que contienen alcaloides, compuestos fenoÌlicos, flavonoides, taninos gaÌlicos y catecoÌlicos. Este estudio deja evidencia del potencial medicinal de las plantas silvestres alto andinas; por lo tanto, los estudios farmacoloÌgicos, fitoquiÌmicos, ecoloÌgicos y biotecnoloÌgicos contribuiriÌan en la promocioÌn de su conservacioÌn y uso sustentable debido a su alta vulnerabilidad y riesgo de extincioÌn.