Aim: Aguas Calientes (AC) is one of the very few geothermal springs located deep into the Amazon ... more Aim: Aguas Calientes (AC) is one of the very few geothermal springs located deep into the Amazon rainforest in Peru. The main aim of this study was to generate an illumina based high resolution microbial phylogenetic profile of the soil-mousse surrounding of an Amazonian geothermal spring like AC. Study Design: Soil-mousse surrounding of AC geothermal spring was subjected to metagenome sequencing using Illumina HiSeq platform. Place and Duration of Study: Soil samples were collected from the surrounding of Aguas Calientes (7°21'12'' S, 75°00'54'' W). The duration of the study was from 2013-2016. Methodology: Metagenomic DNA was extracted from pooled soil samples using PowerSoil ® DNA isolation kit and analyzed at 16S rRNA V3-V4 hypervariable region by amplicon metagenome sequencing on Illumina Hiseq platform. QIIME pipeline was used for 16S RNA detection, clustering Original Research Article Paul et al.; BBJ, 15(1): 1-11, 2016; Article no.BBJ.27519 2 and Operational Taxonomic Units (OTUs) picking followed by Biom file generation and statistical analysis. Functional analysis of 16S amplicons was performed using the default settings of PICRUSt. Results: A total of 72 bacterial phyla and 3 archaeal phyla were detected in AC soil. Proteobacteria (50.09%) was found to be the highest represented phylum among bacterial communities while among archaeal communities Crenarchaeota (0.26%) dominated the sample. More than 50% of the sequences in AC soil were found unidentified/novel at the genus level. A plausible facultative mutualistic relationship was predicted among some members of the communities. In Clusters of Orthologs (COGs) analysis, most of the sequences were found to be associated with " Amino acid transport and metabolism " (8.3%) category, while among predicted Kyoto Encyclopedia of Genes and Genomes (KEGGs) pathways "Membrane transport" (12.1%) was the most abundant one. Conclusion: This is the first report of a high resolution microbial phylogenetic profile of an Amazonian geothermal spring soil-mousse surrounding. Although a very diverse group of the bacterial and archaeal population was observed in the sample, a large portion of unidentified thermophilic microbial members was also noticed which need to be studied.
Aim: Aguas Calientes (AC) is an isolated geothermal spring located deep into the Peruvian Amazon ... more Aim: Aguas Calientes (AC) is an isolated geothermal spring located deep into the Peruvian Amazon rainforest. The principal aim of this study was to isolate and characterize thermophilic lignocellulolytic bacterial strains from AC. Study Design: Bacterial strains were cultured in laboratory and screened for cellulase activity. Superior lignocellulolytic strains were characterized by 16S rRNA sequences. Place and Duration of Study: Samples were collected from Aguas Calientes (7°21'12'' S, 75°00'54'' W). The duration of the study was 2013-20 16. Methodology: Primary isolation of lignocelluloytic bacterial strains was carried out by direct plating, filtration and water enrichment analysis techniques. Secondary screening was performed by semi quantitative plate clearing assay. In both primary and secondary screening assay the growth media contained carboxymethylcellulose (CMC). CMC hydrolysis by bacterial colonies was evaluated by Congo red solution. Selected strains with higher cellulase activity were characterized by 16s rRNA gene sequencing. Results: A total of 22 best performing strains were finally selected from 110 cellulolytic bacterial strains isolated in this study. Thirteen strains were selected from water sample (CW) while 9 selected from soil sample (CS). Water isolate LMB-AC10 showed highest cellulase activity (9.5 U ml-1) at pH 7.4 among all selected strains while LMB-AC3 was found to be the most consistent one (activity was between 6.5 U ml-1 to 7.5 U ml-1) throughout different alkaline pH levels. In CS, the highest cellulase activity (5.2 U ml-1) was showed by strain LMB-SC4 at pH 7.4. Sequence homology analysis of selected bacterial 16S RNA revealed that most of our strains are similar with different reported strains of Geobacillus, more precisely with Geobacillus thermoleovorans and Geobacillus kaustophilus. Conclusion: For the first time several thermophilic lignocellulolytic bacterial strains were isolated from a remote Amazonian geothermal spring and some of them were found very promising.
Aguas Calientes (AC) is an isolated geothermal spring located deep into the Amazon rainforest (7°... more 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.
Neurocysticercosis is an endemic parasitic disease caused by Taenia solium larva. Although the me... more Neurocysticercosis is an endemic parasitic disease caused by Taenia solium larva. Although the mechanism of infection is not completely understood, it is likely driven by proteolytic activity that degrades the intestinal wall to facilitate oncosphere penetration and further infection.
We analyzed the publicly available T. solium EST/DNA library and identified two contigs comprising a full-length cDNA fragment very similar to Echinococcus granulosus Ag5 protein. The T. solium cDNA sequence included a proteolytic trypsin-like-domain in the C-terminal region, and a thrombospondin type-1 adherence-domain in the N-terminal region. Both the trypsin-like and adherence domains were expressed independently as recombinant proteins in bacterial systems. TsAg5 showed marginal trypsin-like activity and high sequence similarity to Ag5. The purified antigens were tested in a Western immunoblot assay to diagnose human neurocysticercosis. The sensitivity of the trypsin-like-domain was 96.36% in patients infected with extraparenchymal cysts, 75.44% in patients infected with multiple cysts, and 39.62% in patients with a single cyst. Specificity was 76.70%. The thrombospondin type-1 adherence-domain was not specific for neurocysticercosis.
There is a great interest for the use of lignocellulolytic enzymes in several industries and in b... more 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. Eve...
The textile industry creates environmental problems due to the release of highly polluting efflue... more The textile industry creates environmental problems due to the release of highly polluting effluents containing substances from different stages of dyeing that are resistant to light, water, and various chemicals, and most of them are difficult to decolorize because of its synthetic origin. The biological degradation of dyes is an economical and environmentally friendly alternative. The aim of this work was to use biofilms of basidiomycete fungi isolated from the Peruvian rainforest for the decolorization of synthetic reactive dyes, considering the advantages of these systems which include better contact with the surrounding medium, resistance to chemical and physical stress, and higher metabolic activity. Among several isolates, two were selected for their capacity of rapid decolorization of several dyes and their biofilm-forming ability. These strains were molecularly identified as Trametes polyzona LMB-TM5 and Ceriporia sp. LMB-TM1 and used in biofilm cultivation for the decolorization of six reactive dyes and textile effluents. Azo dyes were moderately decolorized by both strains, but Remazol Brilliant Blue R (anthraquinone) and Synozol Turquoise Blue HF-G (phthalocyanine) were highly decolorized (97 and 80 %, respectively) by T. polyzona LMB-TM5. Degradation products were found by HPLC analysis. Simulated effluents made of a mixture of six dyes were moderately decolorized by both strains, but a real textile effluent was highly (93 %) decolorized by T. polyzona LMB-TM5. In summary, T. polyzona LMB-TM5 was more efficient than Ceriporia sp. LMB-TM1 for the decolorization of textile dyes and effluents at high initial rates enabling the development of in-plant continuous biofilm processes.
Alkaline cellulases are demanded by the textile industry for several purposes but commercial prep... more 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.
Aim: Aguas Calientes (AC) is one of the very few geothermal springs located deep into the Amazon ... more Aim: Aguas Calientes (AC) is one of the very few geothermal springs located deep into the Amazon rainforest in Peru. The main aim of this study was to generate an illumina based high resolution microbial phylogenetic profile of the soil-mousse surrounding of an Amazonian geothermal spring like AC. Study Design: Soil-mousse surrounding of AC geothermal spring was subjected to metagenome sequencing using Illumina HiSeq platform. Place and Duration of Study: Soil samples were collected from the surrounding of Aguas Calientes (7°21'12'' S, 75°00'54'' W). The duration of the study was from 2013-2016. Methodology: Metagenomic DNA was extracted from pooled soil samples using PowerSoil ® DNA isolation kit and analyzed at 16S rRNA V3-V4 hypervariable region by amplicon metagenome sequencing on Illumina Hiseq platform. QIIME pipeline was used for 16S RNA detection, clustering Original Research Article Paul et al.; BBJ, 15(1): 1-11, 2016; Article no.BBJ.27519 2 and Operational Taxonomic Units (OTUs) picking followed by Biom file generation and statistical analysis. Functional analysis of 16S amplicons was performed using the default settings of PICRUSt. Results: A total of 72 bacterial phyla and 3 archaeal phyla were detected in AC soil. Proteobacteria (50.09%) was found to be the highest represented phylum among bacterial communities while among archaeal communities Crenarchaeota (0.26%) dominated the sample. More than 50% of the sequences in AC soil were found unidentified/novel at the genus level. A plausible facultative mutualistic relationship was predicted among some members of the communities. In Clusters of Orthologs (COGs) analysis, most of the sequences were found to be associated with " Amino acid transport and metabolism " (8.3%) category, while among predicted Kyoto Encyclopedia of Genes and Genomes (KEGGs) pathways "Membrane transport" (12.1%) was the most abundant one. Conclusion: This is the first report of a high resolution microbial phylogenetic profile of an Amazonian geothermal spring soil-mousse surrounding. Although a very diverse group of the bacterial and archaeal population was observed in the sample, a large portion of unidentified thermophilic microbial members was also noticed which need to be studied.
Aim: Aguas Calientes (AC) is an isolated geothermal spring located deep into the Peruvian Amazon ... more Aim: Aguas Calientes (AC) is an isolated geothermal spring located deep into the Peruvian Amazon rainforest. The principal aim of this study was to isolate and characterize thermophilic lignocellulolytic bacterial strains from AC. Study Design: Bacterial strains were cultured in laboratory and screened for cellulase activity. Superior lignocellulolytic strains were characterized by 16S rRNA sequences. Place and Duration of Study: Samples were collected from Aguas Calientes (7°21'12'' S, 75°00'54'' W). The duration of the study was 2013-20 16. Methodology: Primary isolation of lignocelluloytic bacterial strains was carried out by direct plating, filtration and water enrichment analysis techniques. Secondary screening was performed by semi quantitative plate clearing assay. In both primary and secondary screening assay the growth media contained carboxymethylcellulose (CMC). CMC hydrolysis by bacterial colonies was evaluated by Congo red solution. Selected strains with higher cellulase activity were characterized by 16s rRNA gene sequencing. Results: A total of 22 best performing strains were finally selected from 110 cellulolytic bacterial strains isolated in this study. Thirteen strains were selected from water sample (CW) while 9 selected from soil sample (CS). Water isolate LMB-AC10 showed highest cellulase activity (9.5 U ml-1) at pH 7.4 among all selected strains while LMB-AC3 was found to be the most consistent one (activity was between 6.5 U ml-1 to 7.5 U ml-1) throughout different alkaline pH levels. In CS, the highest cellulase activity (5.2 U ml-1) was showed by strain LMB-SC4 at pH 7.4. Sequence homology analysis of selected bacterial 16S RNA revealed that most of our strains are similar with different reported strains of Geobacillus, more precisely with Geobacillus thermoleovorans and Geobacillus kaustophilus. Conclusion: For the first time several thermophilic lignocellulolytic bacterial strains were isolated from a remote Amazonian geothermal spring and some of them were found very promising.
Aguas Calientes (AC) is an isolated geothermal spring located deep into the Amazon rainforest (7°... more 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.
Neurocysticercosis is an endemic parasitic disease caused by Taenia solium larva. Although the me... more Neurocysticercosis is an endemic parasitic disease caused by Taenia solium larva. Although the mechanism of infection is not completely understood, it is likely driven by proteolytic activity that degrades the intestinal wall to facilitate oncosphere penetration and further infection.
We analyzed the publicly available T. solium EST/DNA library and identified two contigs comprising a full-length cDNA fragment very similar to Echinococcus granulosus Ag5 protein. The T. solium cDNA sequence included a proteolytic trypsin-like-domain in the C-terminal region, and a thrombospondin type-1 adherence-domain in the N-terminal region. Both the trypsin-like and adherence domains were expressed independently as recombinant proteins in bacterial systems. TsAg5 showed marginal trypsin-like activity and high sequence similarity to Ag5. The purified antigens were tested in a Western immunoblot assay to diagnose human neurocysticercosis. The sensitivity of the trypsin-like-domain was 96.36% in patients infected with extraparenchymal cysts, 75.44% in patients infected with multiple cysts, and 39.62% in patients with a single cyst. Specificity was 76.70%. The thrombospondin type-1 adherence-domain was not specific for neurocysticercosis.
There is a great interest for the use of lignocellulolytic enzymes in several industries and in b... more 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. Eve...
The textile industry creates environmental problems due to the release of highly polluting efflue... more The textile industry creates environmental problems due to the release of highly polluting effluents containing substances from different stages of dyeing that are resistant to light, water, and various chemicals, and most of them are difficult to decolorize because of its synthetic origin. The biological degradation of dyes is an economical and environmentally friendly alternative. The aim of this work was to use biofilms of basidiomycete fungi isolated from the Peruvian rainforest for the decolorization of synthetic reactive dyes, considering the advantages of these systems which include better contact with the surrounding medium, resistance to chemical and physical stress, and higher metabolic activity. Among several isolates, two were selected for their capacity of rapid decolorization of several dyes and their biofilm-forming ability. These strains were molecularly identified as Trametes polyzona LMB-TM5 and Ceriporia sp. LMB-TM1 and used in biofilm cultivation for the decolorization of six reactive dyes and textile effluents. Azo dyes were moderately decolorized by both strains, but Remazol Brilliant Blue R (anthraquinone) and Synozol Turquoise Blue HF-G (phthalocyanine) were highly decolorized (97 and 80 %, respectively) by T. polyzona LMB-TM5. Degradation products were found by HPLC analysis. Simulated effluents made of a mixture of six dyes were moderately decolorized by both strains, but a real textile effluent was highly (93 %) decolorized by T. polyzona LMB-TM5. In summary, T. polyzona LMB-TM5 was more efficient than Ceriporia sp. LMB-TM1 for the decolorization of textile dyes and effluents at high initial rates enabling the development of in-plant continuous biofilm processes.
Alkaline cellulases are demanded by the textile industry for several purposes but commercial prep... more 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.
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Papers by Marcel Gutiérrez-Correa
We analyzed the publicly available T. solium EST/DNA library and identified two contigs comprising a full-length cDNA fragment very similar to Echinococcus granulosus Ag5 protein. The T. solium cDNA sequence included a proteolytic trypsin-like-domain in the C-terminal region, and a thrombospondin type-1 adherence-domain in the N-terminal region. Both the trypsin-like and adherence domains were expressed independently as recombinant proteins in bacterial systems. TsAg5 showed marginal trypsin-like activity and high sequence similarity to Ag5. The purified antigens were tested in a Western immunoblot assay to diagnose human neurocysticercosis. The sensitivity of the trypsin-like-domain was 96.36% in patients infected with extraparenchymal cysts, 75.44% in patients infected with multiple cysts, and 39.62% in patients with a single cyst. Specificity was 76.70%. The thrombospondin type-1 adherence-domain was not specific for neurocysticercosis.
We analyzed the publicly available T. solium EST/DNA library and identified two contigs comprising a full-length cDNA fragment very similar to Echinococcus granulosus Ag5 protein. The T. solium cDNA sequence included a proteolytic trypsin-like-domain in the C-terminal region, and a thrombospondin type-1 adherence-domain in the N-terminal region. Both the trypsin-like and adherence domains were expressed independently as recombinant proteins in bacterial systems. TsAg5 showed marginal trypsin-like activity and high sequence similarity to Ag5. The purified antigens were tested in a Western immunoblot assay to diagnose human neurocysticercosis. The sensitivity of the trypsin-like-domain was 96.36% in patients infected with extraparenchymal cysts, 75.44% in patients infected with multiple cysts, and 39.62% in patients with a single cyst. Specificity was 76.70%. The thrombospondin type-1 adherence-domain was not specific for neurocysticercosis.