Understanding the Role of Free-Living Bacteria in the Gut of the Lower Termite Coptotermes gestroi Based on Metagenomic DNA Analysis.

Termites' digestive systems, particularly in lower termites with the presence of protozoa, are unique ecological niches that shelter a diverse microbiota with a variety of functions for the host and the environment. In 2012, the metagenomic DNA (5.4 Gb) of the prokaryotes that freely live in the gut of the lower termite Coptotermes gestroi were sequenced. A total of 125,431 genes were predicted and analyzed in order to mine lignocellulolytic genes. however, the overall picture of the structure, diversity, and function of the prokaryotic gut microbiota was not investigated. In the present study, these 125,431 genes were taxonomically classified by MEGAN and functionally annotated by the Kyoto Encyclopedia of Genes and Genomes (KEGG) and by the Carbohydrate-Active enZYmes (CAZy) and HMMER databases. As a result, 95,751 bacterial genes were classified into 35 phyla. The structure of the bacteria, typified by a high ratio of Firmicutes to Bacterioidetes, was distinct from the structure of the entirety of the bacteria in the lower or higher termites' guts. The archaea (533 genes) were distributed into 4 phyla, 10 classes, 15 orders, 21 families, 47 genera, and 61 species. Although freely living in the guts, the prokaryotic community was formed, developed, and adapted to exhibit unique interactions in order to perform mutual roles of benefit to their hosts. Methanobacteriales, accounting for 61% of the archaea symbionts, seem to play an important role in methanogenesis. Concomitantly, bacterial methanotrophs in the gut utilize methane and combine with other bacterial groups, including potential lignocellulolytic degraders, acetogens, sulfur bacteria, and nitrogen-recycling bacteria, to efficiently convert wood with little nitrogen into acetates via certain pathway modules specified by prokaryotes that freely live in the gut. This forms an important energy source for the termites. Furthermore, bacteria carry 2223 genes involved in the biosynthesis of 17 antibiotic groups. The gut bacteria also possess genes for the degradation of 18 toxic aromatic compounds, of which four are commercial pesticides against termites commonly used for the preservation of wooden constructions. Eight of the eighteen pathways were the first to be reported from the termite gut. Overall, this study sheds light on the roles of the freely living bacteria and archaea in the C. gestroi gut, providing evidence that the gut microbiome acts as the second host genome, contributing both nutrients and immunity to support the host's existence, growth, and development.

Extensive comparison of methods for removal of organic halogen compounds from pharmaceutical process wastewaters with life cycle, PESTLE, and multi-criteria decision analyses.

Recycling and disposing wastewater from the pharmaceutical industry are of utmost importance in mitigating chemical waste generation, where unmanaged hazardous waste fluxes could cause massive environmental damage. Air stripping, steam stripping, distillation, and incineration offer significant emission reduction potentials for pharmaceutical applications; however, selecting specific process units is a complicated task due to the high number of influencing screening criteria. The mentioned chemical processes are modelled with the Aspen Plus program. This study examines the environmental impacts of adsorbable organic halogens (AOX) containing pharmaceutical process wastewater disposal by conducting life cycle impact assessments using the Product Environmental Footprint (PEF), IMPACT World + Endpoint V1.01, and Recipe 2016 Endpoint (H) V1.06 methods. The results show that the distillation-based separation of AOX compounds is characterized by the most favourable climate change impact and outranks the PEF single score of air stripping, steam stripping, and incineration by 6.3%, 29.1%, 52.0%, respectively. The energy-intensive distillation technology is further evaluated by considering a wide selection of energy sources (i.e., fossil fuel, nuclear, solar, wind onshore, and wind offshore) using PESTLE (Political, Economic, Social, Technological, Legal, Environmental) analysis combined with multi-criteria decision support to determine the most beneficial AOX disposal scenario. The best overall AOX regeneration performance and lowest climate change impact (7.25 × 10-3 kg CO2-eq (1 kg purified wastewater)-1) are obtained by supplying variable renewable electricity from onshore wind turbines, reaching 64.87% carbon emission reduction compared to the baseline fossil fuel-based process alternative.

Life Cycle, PESTLE and Multi-Criteria Decision Analysis of Membrane Contactor-Based Nitrogen Recovery Process.

Nitrogen is one of the most critical nutrients in the biosphere, and it is an essential nutrient for plant growth. Nitrogen exists in the atmosphere vastly as a gaseous form, but only reactive nitrogen is usable for plants. It is a valuable resource and worth recovering in the wastewater sector. The aim of this work was to prepare a comprehensive environmental analysis of a novel membrane contactor-based process, which is capable of highly efficient nitrogen removal from wastewater. Life cycle assessment (LCA), PESTLE and multi-criteria decision analysis (MCDA) were applied to evaluate the process. The EF 3.0 method, preferred by the European Commission, IMPACT World+, ReCiPe 2016 and IPCC 2021 GWP100 methods were used with six different energy resources-electricity high voltage, solar, nuclear, heat and power and wind energy. The functional unit of 1 m3 of water product was considered as output and "gate-to-gate" analysis was examined. The results of our study show that renewable energy resources cause a significantly lower environmental load than traditional energy resources. TOPSIS score was used to evaluate the alternatives in the case of MCDA. For the EU region, the most advantageous option was found to be wind energy onshore with a score of 0.76, and the following, nuclear, was 0.70.

Understanding the Role of Prevotella Genus in the Digestion of Lignocellulose and Other Substrates in Vietnamese Native Goats' Rumen by Metagenomic Deep Sequencing.

Bacteria in rumen play pivotal roles in the digestion of nutrients to support energy for the host. In this study, metagenomic deep sequencing of bacterial metagenome extracted from the goats' rumen generated 48.66 GB of data with 3,411,867 contigs and 5,367,270 genes. The genes were mainly functionally annotated by Kyoto Encyclopedia of Genes and Genomes (KEGG) Carbohydrate-Active enZYmes (CAZy), and HMMER database, and taxonomically classified by MEGAN. As a result, 65,554 genes encoding for 30 enzymes/proteins related to lignocellulose conversion were exploited, in which nine enzymes were seen for the first time in goat rumen. Prevotella was the most abundant genus, contributing 30% hemicellulases and 36% enzymes/proteins for lignocellulose pretreatment, and supporting 98.8% of feruloyl esterases and 71.7% acetylxylan esterases. In addition, 18 of the 22 most lignocellulose digesting- potential contigs belonged to Prevotella. Besides, Prevotella possessed many genes coding for amylolytic enzymes. One gene encoding for endoxylanase was successfully expressed in E. coli. The recombinant enzyme had high Vmax, was tolerant to some salts and detergents, worked better at pH 5.5-6.5, temperature 40-50 °C, and was capable to be used in practices. Based on these findings, we confirm that Prevotella plays a pivotal role for hemicellulose digestion and significantly participates in starch, cellulose, hemicellulose, and pectin digestion in the goat rumen.

Some characters of bacterial cellulases in goats' rumen elucidated by metagenomic DNA analysis and the role of fibronectin 3 module for endoglucanase function.

OBJECTIVE: Fibronectin 3 (FN3) and immunoglobulin like modules (Ig) are usually collocated beside modular cellulase catalytic domains. However, very few researches have investigated the role of these modules. In a previous study, we have sequenced and analyzed bacterial metagenomic DNA in Vietnamese goats' rumen and found that cellulase-producing bacteria and cellulase families were dominant. In this study, the properties of modular cellulases and the role of a FN3 in unique endoglucanase belonging to glycosyl hydorlase (GH) family 5 were determined. METHODS: Based on Pfam analysis, the cellulases sequences containing FN3, Ig modules were extracted from 297 complete open reading frames (ORFs). The alkaline, thermostability, tertiary structure of deduced enzymes were predicted by AcalPred, TBI software, Phyre2 and Swiss models. Then, whole and truncated forms of a selected gene were expressed in Escherichia coli and purified by His-tag affinity column for assessment of FN3 ability to enhance enzyme activity, solubility and conformation. RESULTS: From 297 complete ORFs coding for cellulases, 148 sequences containing FN3, Ig were identified. Mostly FN3 appeared in 90.9% beta-glucosidases belonging to glycosyl hydrolase family 3 (GH3) and situated downstream of catalytic domains. The Ig was found upstream of 100% endoglucanase GH9. Rarely FN3 was seen to be situated downstream of X domain and upstream of catalytic domain endoglucanase GH5. Whole enzyme (called XFN3GH5 based on modular structure) and truncate forms FN3, XFN3, FN3GH5, GH5 were cloned in pET22b (+) and pET22SUMO to be expressed in single and fusion forms with a small ubiquitin-related modifier partner (S). The FN3, SFN3 increased GH5 solubility in FN3GH5, SFN3GH5. The SFN3 partly served for GH5 conformation in SFN3GH5, increased modules interaction and enzyme-soluble substrate affinity to enhance SXFN3GH5, SFN3GH5 activities in mixtures. Both SFN3 and SXFN3 did not anchor enzyme on filter paper but exfoliate and separate cellulose chains on filter paper for enzyme hydrolysis. CONCLUSION: Based on these findings, the presence of FN3 module in certain cellulases was confirmed and it assisted for enzyme conformation and activity in both soluble and insoluble substrate.

Separation of Alcohol-Water Mixtures by a Combination of Distillation, Hydrophilic and Organophilic Pervaporation Processes.

It can be stated that in the fine chemical industries, especially in the pharmaceutical industry, large amounts of liquid waste and industrial waste solvents are generated during the production technology. Addressing these is a key issue because their disposal often accounts for the largest proportion of the cost of the entire technology. There is need to develop regeneration processes that are financially beneficial to the plant and, if possible, reuse the liquid waste in the spirit of a circular economy, in a particular technology, or possibly elsewhere. The distillation technique proves to be a good solution in many cases, but in the case of mixtures with high water content and few volatile components, this process is often not cost-effective due to its high steam consumption, and in the case of azeotropic mixtures there are separation constraints. In the present work, the membrane process considered as an alternative; pervaporation is demonstrated through the treatment of low alcohol (methanol and ethanol) aqueous mixtures. Alcohol-containing process wastewaters were investigated in professional process simulator environment with user-added pervaporation modules. Eight different methods were built up in ChemCAD flowsheet simulator: organophilic pervaporation (OPV), hydrophilic pervaporation (HPV), hydrophilic pervaporation with recirculation (R-HPV), dynamic organophilic pervaporation (Dyn-OPV), dynamic hydronophilic pervaporation (Dyn-HPV), hybrid distillation-organophilic pervaporation (D + OPV), hybrid distillation-hydrophilic pervaporation (D + HPV), and finally hybrid distillation-hydrophilic pervaporation with recirculation (R-D + HPV). It can be stated the last solution in line was the most suitable in the terms of composition, however distillation of mixture with high water content has significant heat consumption. Furthermore, the pervaporation supplemented with dynamic tanks is not favourable due to the high recirculation rate in the case of tested mixtures and compositions.

Adipose tissue-derived cytokines and their correlations with clinical characteristics in Vietnamese patients with type 2 diabetes mellitus.

BACKGROUND: Adipokines are involved in the pathogenesis of metabolic disorders including obesity and type 2 diabetes mellitus (T2DM). This study investigates the levels of leptin, resistin, visfatin, secreted frizzled-related protein 5 (SFRP5), monocyte chemoattractant protein-1 (MCP-1) and retinol-binding protein 4 (RBP4) and their correlations with clinical parameters of overweight and T2DM. METHODS: We recruited overweight 50 patients with T2DM, 88 non-overweight patients with T2DM, 29 overweight and 100 non-overweight individuals devoid of T2DM for this study. The levels of studied adipokines were measured by enzyme-linked immunosorbent assay and correlated with clinical parameters. RESULTS: The levels of MCP-1 and SFRP5 were decreased while visfatin and RBP4 levels were increased in patients with T2DM compared to those in the control individuals (P < 0.01). Among patients with T2DM, leptin and resistin levels were higher while RBP4 levels were lower in patients with overweight T2DM compared to those in patients with non-overweight T2DM (P < 0.0001, 0.019 and 0.05, respectively). Leptin and MCP-1 levels were correlated with HOMA-IR, QUICKI and HOMA-ß. Leptin/MCP-1 ratio was correlated with insulin levels, HOMA-IR and HOMA-ß indexes. Resistin/RBP4, visfatin/MCP-1 and MCP-1/RBP4 ratios were strongly correlated with the levels of fasting glucose, HbA1c and HOMA-ß. In addition, ROC curve analyses indicated a diagnostic potential of resistin/RBP4 and MCP-1/RBP4 indexes for T2DM (AUC = 0.81 and 0.83, respectively) and ß-cell function (AUC = 0.76 and 0.74, respectively). CONCLUSIONS: Adipokines (leptin, resistin, visfatin, SFRP5, MCP-1, and RBP4) are associated with overweight and T2DM and may serve as a potential prognostic marker and therapeutic intervention for overweight-related T2DM.

Metagenomic insights into lignocellulose-degrading genes through Illumina-based de novo sequencing of the microbiome in Vietnamese native goats' rumen.

The scarcity of enzymes having an optimal activity in lignocellulose deconstruction is an obstacle for industrial-scale conversion of cellulosic biomass into biofuels. With the aim of mining novel lignocellulolytic enzymes, a ~9 Gb metagenome of bacteria in Vietnamese native goats' rumen was sequenced by Illumina platform. From the data, 821 ORFs encoding carbohydrate esterases (CEs) and polysaccharide lyases (PLs) serving for lignocellulose pre-treatment, 816 ORFs encoding 11 glycoside hydrolase families (GHs) of cellulases, and 2252 ORFs encoding 22 GHs of hemicellulases, were mined. The carbohydrate binding module (CBM) was also abundant with 763 ORFs, of which 480 ORFs are located with lignocellulolytic enzymes. The enzyme modularity analysis showed that CBMs are usually present in endoglucanase, endo 1,3-beta-D-glucosidase, and endoxylanase, whereas fibronectin 3-like module (FN3) mainly represents in GH3 and immunoglobulin-like domain (Ig) was located in GH9 only. Every domain located in each ORF was analyzed in detail to contribute enzymes' modularity which is valuable for modelling, to study the structure, and for recombinant production. With the aim of confirming the annotated results, a mined ORF encoding CBM63 was highly expressed in E. coli in soluble form. The purified recombinant CBM63 exhibited no cellulase activity, but enhanced a commercial cellulase activity in the destruction of a paper filter.

Metagenomic analysis of bacterial community structure and diversity of lignocellulolytic bacteria in Vietnamese native goat rumen.

OBJECTIVE: In a previous study, analysis of Illumina sequenced metagenomic DNA data of bacteria in Vietnamese goats' rumen showed a high diversity of putative lignocellulolytic genes. In this study, taxonomy speculation of microbial community and lignocellulolytic bacteria population in the rumen was conducted to elucidate a role of bacterial structure for effective degradation of plant materials. METHODS: The metagenomic data had been subjected into Basic Local Alignment Search Tool (BLASTX) algorithm and the National Center for Biotechnology Information non-redundant sequence database. Here the BLASTX hits were further processed by the Metagenome Analyzer program to statistically analyze the abundance of taxa. RESULTS: Microbial community in the rumen is defined by dominance of Bacteroidetes compared to Firmicutes. The ratio of Firmicutes versus Bacteroidetes was 0.36:1. An abundance of Synergistetes was uniquely identified in the goat microbiome may be formed by host genotype. With regard to bacterial lignocellulose degraders, the ratio of lignocellulolytic genes affiliated with Firmicutes compared to the genes linked to Bacteroidetes was 0.11:1, in which the genes encoding putative hemicellulases, carbohydrate esterases, polysaccharide lyases originated from Bacteroidetes were 14 to 20 times higher than from Firmicutes. Firmicutes seem to possess more cellulose hydrolysis capacity showing a Firmicutes/Bacteroidetes ratio of 0.35:1. Analysis of lignocellulolytic potential degraders shows that four species belonged to Bacteroidetes phylum, while two species belonged to Firmicutes phylum harbouring at least 12 different catalytic domains for all lignocellulose pretreatment, cellulose, as well as hemicellulose saccharification. CONCLUSION: Based on these findings, we speculate that increasing the members of Bacteroidetes to keep a low ratio of Firmicutes versus Bacteroidetes in goat rumen has resulted most likely in an increased lignocellulose digestion.

Improved cell surface display of Salmonella enterica serovar Enteritidis antigens in Escherichia coli.

BACKGROUND: Salmonella enterica serovar Enteritidis (SE) is one of the most potent pathogenic Salmonella serotypes causing food-borne diseases in humans. We have previously reported the use of the ß-autotransporter AIDA-I to express the Salmonella flagellar protein H:gm and the SE serotype-specific fimbrial protein SefA at the surface of E. coli as live bacterial vaccine vehicles. While SefA was successfully displayed at the cell surface, virtually no full-length H:gm was exposed to the medium due to extensive proteolytic cleavage of the N-terminal region. In the present study, we addressed this issue by expressing a truncated H:gm variant (H:gmd) covering only the serotype-specific central region. This protein was also expressed in fusion to SefA (H:gmdSefA) to understand if the excellent translocation properties of SefA could be used to enhance the secretion and immunogenicity. RESULTS: H:gmd and H:gmdSefA were both successfully translocated to the E. coli outer membrane as full-length proteins using the AIDA-I system. Whole-cell flow cytometric analysis confirmed that both antigens were displayed and accessible from the extracellular environment. In contrast to H:gm, the H:gmd protein was not only expressed as full-length protein, but it also seemed to promote the display of the protein fusion H:gmdSefA. Moreover, the epitopes appeared to be recognized by HT-29 intestinal cells, as measured by induction of the pro-inflammatory interleukin 8. CONCLUSIONS: We believe this study to be an important step towards a live bacterial vaccine against Salmonella due to the central role of the flagellar antigen H:gm and SefA in Salmonella infections and the corresponding immune responses against Salmonella.

Mining biomass-degrading genes through Illumina-based de novo sequencing and metagenomic analysis of free-living bacteria in the gut of the lower termite Coptotermes gestroi harvested in Vietnam.

The 5.6 Gb metagenome of free-living microbial flora in the gut of the lower termite Coptotermes gestroi, harvested in Vietnam, was sequenced using Illumina technology. Genes related to biomass degradation were mined for a better understanding of biomass digestion in the termite gut and to identify lignocellulolytic enzymes applicable to biofuel production. The sequencing generated 5.4 Gb of useful reads, containing 125,431 ORFs spanning 78,271,365 bp, 80% of which was derived from bacteria. The 12 most abundant bacterial orders were Spirochaetales, Lactobacillales, Bacteroidales, Clostridiales, Enterobacteriales, Pseudomonades, Synergistales, Desulfovibrionales, Xanthomonadales, Burkholderiales, Bacillales, and Actinomycetales, and 1460 species were estimated. Of more than 12,000 ORFs with predicted functions related to carbohydrate metabolism, 587 encoding hydrolytic enzymes for cellulose, hemicellulose, and pectin were identified. Among them, 316 ORFs were related to cellulose degradation, and included ß-glucosidases, 6-phospho-ß-glucosidases, licheninases, glucan endo-1,3-ß-D-glucosidases, endoglucanases, cellulose 1,4-ß-cellobiosidases, glucan 1,3-ß-glucosidases, and cellobiose phosphorylases. In addition, 259 ORFs were related to hemicellulose degradation, encoding endo-1,4-ß-xylanases, α-galactosidases, α-N-arabinofuranosidases, xylan 1,4-ß-xylosidases, arabinan endo-1,5-α-L-arabinosidases, endo-1,4-ß-mannanases, and α-glucuronidases. Twelve ORFs encoding pectinesterases and pectate lyases were also obtained. To our knowledge, this is the first successful application of Illumina-based de novo sequencing for the analysis of a free-living bacterial community in the gut of a lower termite C. gestroi and for mining genes related to lignocellulose degradation from the gut bacteria.

A dual tag system for facilitated detection of surface expressed proteins in Escherichia coli.

BACKGROUND: The discovery of the autotransporter family has provided a mechanism for surface expression of proteins in laboratory strains of Escherichia coli. We have previously reported the use of the AIDA-I autotransport system to express the Salmonella enterica serovar Enteritidis proteins SefA and H:gm. The SefA protein was successfully exposed to the medium, but the orientation of H:gm in the outer membrane could not be determined due to proteolytic cleavage of the N-terminal detection-tag. The goal of the present work was therefore to construct a vector containing elements that facilitates analysis of surface expression, especially for proteins that are sensitive to proteolysis or otherwise difficult to express. RESULTS: The surface expression system pAIDA1 was created with two detection tags flanking the passenger protein. Successful expression of SefA and H:gm on the surface of E. coli was confirmed with fluorescently labeled antibodies specific for the N-terminal His6-tag and the C-terminal Myc-tag. While both tags were detected during SefA expression, only the Myc-tag could be detected for H:gm. The negative signal indicates a proteolytic cleavage of this protein that removes the His6-tag facing the medium. CONCLUSIONS: Expression levels from pAIDA1 were comparable to or higher than those achieved with the formerly used vector. The presence of the Myc- but not of the His6-tag on the cell surface during H:gm expression allowed us to confirm the hypothesis that this fusion protein was present on the surface and oriented towards the cell exterior. Western blot analysis revealed degradation products of the same molecular weight for SefA and H:gm. The size of these fragments suggests that both fusion proteins have been cleaved at a specific site close to the C-terminal end of the passenger. This proteolysis was concluded to take place either in the outer membrane or in the periplasm. Since H:gm was cleaved to a much greater extent then the three times smaller SefA, it is proposed that the longer translocation time for the larger H:gm makes it more susceptible to proteolysis.

Mutations suppressing the loss of DegQ function in Bacillus subtilis (natto) poly-γ-glutamate synthesis.

The degQ gene of Bacillus subtilis (natto), encoding a small peptide of 46 amino acids, is essential for the synthesis of extracellular poly-gamma-glutamate (γPGA). To elucidate the role of DegQ in γPGA synthesis, we knocked out the degQ gene in Bacillus subtilis (natto) and screened for suppressor mutations that restored γPGA synthesis in the absence of DegQ. Suppressor mutations were found in degS, the receptor kinase gene of the DegS-DegU two-component system. Recombinant DegS-His(6) mutant proteins were expressed in Escherichia coli cells and subjected to an in vitro phosphorylation assay. Compared with the wild type, mutant DegS-His(6) proteins showed higher levels of autophosphorylation (R208Q, M195I, L248F, and D250N), reduced autodephosphorylation (D250N), reduced phosphatase activity toward DegU, or a reduced ability to stimulate the autodephosphorylation activity of DegU (R208Q, D249G, M195I, L248F, and D250N) and stabilized DegU in the phosphorylated form. These mutant DegS proteins mimic the effect of DegQ on wild-type DegSU in vitro. Interestingly, DegQ stabilizes phosphorylated DegS only in the presence of DegU, indicating a complex interaction of these three proteins.

Expression of the pgsB encoding the poly-gamma-DL-glutamate synthetase of Bacillus subtilis (natto).

An industrial strain of Bacillus subtilis (natto) was used to produce poly-gamma-DL-glutamate (gammaPGA), a polymer of DL-glutamate linked by a gamma-peptide bond. In spite of efforts to improve gammaPGA production by modifying the medium, little attention has been paid to the expression of the gammaPGA synthetase gene. In this study, we investigated the expression of the gammaPGA synthetic gene and the gammaPGA product under various conditions with the LacZ-fusion of the synthetic gene (pgsB-lacZ). The 5' upstream regulatory region of the pgsB gene was also investigated by constructing deletion mutations of lacZ-fusion. The pgsB-lacZ was clearly expressed in the early stationary phase and was abolished by degU gene disruption. The results showed that pgsB-lacZ expression was repressed in rich media, and that gammaPGA production was limited by the substrate supply rather than by the amount of synthetase. Adding D-glutamate to the medium reduced gammaPGA production and synthetic gene expression. The transcription start point was determined by primer extension, and it was found that up to -721 bp (translation start point = +1) of the 5' untranslated region (UTR) was required for optimal pgsB-lacZ fusion gene expression.