Microbial degradation and community structure analysis of hydroxyl-terminated polybutadiene (HTPB)

Hydroxyl-terminated polybutadiene (HTPB) is a curing adhesive that is commonly used in the production of ammunition, and it emerged during the time of war. After entering the peaceful era, several countries around the globe have focused on the destruction of expired ammunition using safe and economical methods in terms of consumption of energy. Microorganisms exhibit a highly efficient and environment friendly degradation capability for variety of refractory substances. Therefore, in this study we screened five strains of microorganisms from five environmental soil samples for their ability to degrade HTPB. These microorganisms were identified as Microbacterium trichothecenolyticum, Microbacterium esteraromaticum, Arthrobacter pascens, Pseudonocardia carboxydivorans and Ochrobactrum anthropic based on 16S rRNA gene similarity index. We observed the uncorroded and corroded HTPB sample through scanning electron microscopy and observed the formation of lot of holes and gullies in HTPB after corrosion. An 18S rRNA gene clone library was constructed for HTPB-degrading fungi. Based on the results of library evaluation, it was found that the structure of the HTPB-degrading fungi community was relatively simple. A total of 54 positive clones were obtained. These clones represented some uncultured microorganisms that were closely related to Scytalidium lignicola, Pseudokahliella and Gonostomum strenuum. This study will help in the implementation of environment friendly degradation strategies for HTPB degradation.

In silico characterization of the GH5-cellulase family from uncultured microorganisms: physicochemical and structural studies

Background Hydrolysis of cellulose-based biomass by cellulases produce fermented sugar for making biofuels, such as bioethanol. Cellulases hydrolyze the β-1,4-glycosidic linkage of cellulose and can be obtained from cultured and uncultured microorganisms. Uncultured microorganisms are a source for exploring novel cellulase genes through the metagenomic approach. Metagenomics concerns the extraction, cloning, and analysis of the entire genetic complement of a habitat without cultivating microbes. The glycoside hydrolase 5 family (GH5) is a cellulase family, as the largest group of glycoside hydrolases. Numerous variants of GH5-cellulase family have been identified through the metagenomic approach, including CelGH5 in this study. University-CoE-Research Center for Biomolecule Engineering, Universitas Airlangga successfully isolated CelGH5 from waste decomposition of oil palm empty fruit bunches (OPEFB) soil by metagenomics approach. The properties and structural characteristics of GH5-cellulases from uncultured microorganisms can be studied using computational tools and software. Results The GH5-cellulase family from uncultured microorganisms was characterized using standard computational-based tools. The amino acid sequences and 3D-protein structures were retrieved from the GenBank Database and Protein Data Bank. The physicochemical analysis revealed the sequence length was roughly 332–751 amino acids, with the molecular weight range around 37–83 kDa, dominantly negative charges with pI values below 7. Alanine was the most abundant amino acid making up the GH5-cellulase family and the percentage of hydrophobic amino acids was more than hydrophilic. Interestingly, ten endopeptidases with the highest average number of cleavage sites were found. Another uniqueness demonstrated that there was also a difference in stability between in silico and wet lab. The II values indicated CelGH5 and ACA61162.1 as unstable enzymes, while the wet lab showed they were stable at broad pH range. The program of SOPMA, PDBsum, ProSA, and SAVES provided the secondary and tertiary structure analysis. The predominant secondary structure was the random coil, and tertiary structure has fulfilled the structure quality of QMEAN4, ERRAT, Ramachandran plot, and Z score. Conclusion This study can afford the new insights about the physicochemical and structural properties of the GH5-cellulase family from uncultured microorganisms. Furthermore, in silico analysis could be valuable in selecting a highly efficient cellulases for enhanced enzyme production.

Study of enterobacteria biofilms critical point control technologies livestock and food production

The relevance of the study and the presence of gaps in the existing knowledge on the topic. Monitoring studies of the biological safety of food raw materials for microbiological indicators is an urgent problem due to the increase in the number registered diseases transmitted to humans through raw materials and products of animal origin. There is a tendency for a statistically significant increase in epidemiological indicators throughout the world, the proportion of these pathologies is increasing both in humane medicine and veterinary medicine. The aim of the work is a comparative assessment and selection of effective methods for studying the formation biofilms enterobacteriaceae circulating among susceptible animal species and isolated from food raw materials.Methods. Аnalysis of growth and dynamics development biofilms Enterobacteriaceae was carried out during cultivation on nutrient media containing growth factors for the repair of the cell wall and the reversal viable uncultured microorganisms. To study the morphological and functional patterns of the development a population microorganisms in vitro and in vivo, we used the conventional and developed methods for preparing preparations for scanning, transmission phase contrast, optical and luminescence microscopy.Results and its discussion. During microbiological control critical points in the technology of animal husbandry and food production, the morphological and functional characteristics biofilms, which are communities microorganisms secreting a polymer matrix and adhered to the tissues of susceptible animal species and abiotic surfaces livestock buildings and food industries, were studied. The developed methods of biofilm cultivation made it possible to study enterobacteriaceae biofilms in vitro and in vivo, without disturbing the natural architectonics of the population microorganisms, to determine the components extracellular matrix. For the study dynamics morphological and functional patterns of the development populations microorganisms, routine and technological advances present are recognized as promising, for example, scanning electron microscopy makes it possible to assess the degree of formation and morphological composition biofilms. Phase contrast microscopy to reveal processes depending on the composition medium and the oxygen content in the culture medium.Conclusions. Methods for cultivating biofilms in vitro and in vivo without disturbing the natural architectonics of biofilms made it possible to optimize the preparation samples for research and eliminate the routine stages of colony counting, and significantly increase the number of analyzes. Due to the simplicity of operations and minimization manual labor, productivity increases, safety of work is increased, cost of personnel working time is reduced, and subjective factors are excluded. For the development of a complex antiepizootic and diagnostic measures, a priority direction is the disclosure of scientific knowledge in the field fundamental studies of ecological plasticity and adaptation potentially pathogenic enterobacteria to parasitism in the warm-blooded organism of birds and mammals. This will allow solving applied problems controlling the critical points of livestock and food production technology, developing effective chemotherapeutic and disinfecting drugs to reduce cell coaggregation and detect viable uncultured microorganisms.

Novel clades of soil biphenyl degraders revealed by integrating isotope probing, multi-omics, and single-cell analyses

Most microorganisms in the biosphere remain uncultured and poorly characterized. Although the surge in genome sequences has enabled insights into the genetic and metabolic properties of uncultured microorganisms, their physiology and ecological roles cannot be determined without direct probing of their activities in natural habitats. Here we employed an experimental framework coupling genome reconstruction and activity assays to characterize the largely uncultured microorganisms responsible for aerobic biodegradation of biphenyl as a proxy for a large class of environmental pollutants, polychlorinated biphenyls. We used 13C-labeled biphenyl in contaminated soils and traced the flow of pollutant-derived carbon into active cells using single-cell analyses and protein–stable isotope probing. The detection of 13C-enriched proteins linked biphenyl biodegradation to the uncultured Alphaproteobacteria clade UBA11222, which we found to host a distinctive biphenyl dioxygenase gene widely retrieved from contaminated environments. The same approach indicated the capacity of Azoarcus species to oxidize biphenyl and suggested similar metabolic abilities for species of Rugosibacter. Biphenyl oxidation would thus represent formerly unrecognized ecological functions of both genera. The quantitative role of these microorganisms in pollutant degradation was resolved using single-cell-based uptake measurements. Our strategy advances our understanding of microbially mediated biodegradation processes and has general application potential for elucidating the ecological roles of uncultured microorganisms in their natural habitats.

Strategies for Natural Products Discovery from Uncultured Microorganisms

Microorganisms are highly regarded as a prominent source of natural products that have significant importance in many fields such as medicine, farming, environmental safety, and material production. Due to this, only tiny amounts of microorganisms can be cultivated under standard laboratory conditions, and the bulk of microorganisms in the ecosystems are still unidentified, which restricts our knowledge of uncultured microbial metabolism. However, they could hypothetically provide a large collection of innovative natural products. Culture-independent metagenomics study has the ability to address core questions in the potential of NP production by cloning and analysis of microbial DNA derived directly from environmental samples. Latest advancements in next generation sequencing and genetic engineering tools for genome assembly have broadened the scope of metagenomics to offer perspectives into the life of uncultured microorganisms. In this review, we cover the methods of metagenomic library construction, and heterologous expression for the exploration and development of the environmental metabolome and focus on the function-based metagenomics, sequencing-based metagenomics, and single-cell metagenomics of uncultured microorganisms.

Microdroplet-based system for culturing of environmental microorganisms using FNAP-sort

Droplet microfluidics has emerged as a powerful technology for improving the culturing efficiency of environmental microorganisms. However, its widespread adoption has been limited due to considerable technical challenges, especially related to identification and manipulation of individual growth-positive droplets. Here, we combined microfluidic droplet technology with on-chip “fluorescent nucleic acid probe in droplets for bacterial sorting” (FNAP-sort) for recovery of growth-positive droplets and droplet microdispensing to establish an end-to-end workflow for isolation and culturing of environmental microbes. As a proof-of-concept, we demonstrate the ability of our technique to yield high-purity cultures of rare microorganisms from a representative complex environmental microbiome. As our system employs off-the-shelf commercially available equipment, we believe that it can be readily adopted by others and may thus find widespread use toward culturing the high proportion of as-of-yet uncultured microorganisms in different biomes.

Agarose gel microcapsules enable easy-to-prepare, picolitre-scale single-cell genomics, yielding near-complete genome sequences

A novel type of agarose gel microcapsule (AGM), consisting of an alginate picolitre sol core and an agarose gel shell, was developed to obtain high-quality single-cell amplified genomic DNA of bacteria. The AGM is easy to prepare in a stable emulsion with oil of water-equivalent density which prevents AGM aggregation, with only standard laboratory equipment. Single cells from a pure culture of Escherichia coli, a mock community comprising 15 strains of human gut bacteria, and a termite gut bacterial community were encapsulated within AGMs, and their genomic DNAs were obtained with massively parallel amplifications in a tube. The genome sequencing did not need second-round amplification, and showed an average genome completeness that was much higher than that obtained by the conventional amplification method in microlitre scale, regardless of the genomic guanine-cytosine contents. Our novel method using AGMs allows many researchers to perform single-cell genomics easily and effectively, and can enhance the genome analysis of yet-uncultured microorganisms.

Characterization of efficient xylanases from industrial-scale pulp and paper wastewater treatment microbiota

Xylanases are widely used enzymes in the food, textile, and paper industries. Most efficient xylanases have been identified from lignocellulose-degrading microbiota, such as the microbiota of the cow rumen and the termite hindgut. Xylanase genes from efficient pulp and paper wastewater treatment (PPWT) microbiota have been previously recovered by metagenomics, assigning most of the xylanase genes to the GH10 family. In this study, a total of 40 GH10 family xylanase genes derived from a certain PPWT microbiota were cloned and expressed in Escherichia coli BL21 (DE3). Among these xylanase genes, 14 showed xylanase activity on beechwood substrate. Two of these, PW-xyl9 and PW-xyl37, showed high activities, and were purified to evaluate their xylanase properties. Values of optimal pH and temperature for PW-xyl9 were pH 7 and 60 ℃, respectively, while those for PW-xyl37 were pH 7 and 55 ℃, respectively; their specific xylanase activities under optimal conditions were 470.1 U/mg protein and 113.7 U/mg protein, respectively. Furthermore, the Km values of PW-xyl9 and PW-xyl37 were determined as 8.02 and 18.8 g/L, respectively. The characterization of these two xylanases paves the way for potential application in future pulp and paper production and other industries, indicating that PPWT microbiota has been an undiscovered reservoir of efficient lignocellulase genes. This study demonstrates that a metagenomic approach has the potential to screen efficient xylanases of uncultured microorganisms from lignocellulose-degrading microbiota. In a similar way, other efficient lignocellulase genes might be identified from PPWT treatment microbiota in the future.