scholarly journals Humin Assists Reductive Acetogenesis in Absence of Other External Electron Donor

2020 ◽  
Vol 17 (12) ◽  
pp. 4211 ◽  
Author(s):  
Mahasweta Laskar ◽  
Takuya Kasai ◽  
Takanori Awata ◽  
Arata Katayama
Keyword(s):  
Carbon Source ◽  
Electron Donor ◽  
16S Rrna Genes ◽  
Reductive Dehalogenation ◽  
Rrna Genes ◽  
Extracellular Electron Transfer ◽  
Metagenomic Sequencing ◽  
Electron Mediator ◽  
Total Electron ◽  
Methanogenic Activity

The utilization of extracellular electron transfer by microorganism is highly engaging for remediation of toxic pollutants under “energy-starved” conditions. Humin, an organo-mineral complex of soil, has been instrumental as an external electron mediator for suitable electron donors in the remediative works of reductive dehalogenation, denitrification, and so forth. Here, we report, for the first time, that humin assists microbial acetogenesis as the extracellular electron donor using the electron acceptor CO 2 . Humin was obtained from Kamajima paddy soil, Japan. The anaerobic acetogenic consortium in mineral medium containing CO 2 / HCO 3 − as the inorganic carbon source used suspended humin as the energy source under mesophilic dark conditions. Retardation of acetogenesis under the CO 2 -deficient conditions demonstrated that humin did not function as the organic carbon source but as electron donor in the CO 2 -reducing acetogenesis. The consortium with humin also achieved anaerobic dechlorination with limited methanogenic activity. Total electron-donating capacity of humin was estimated at about 87 µeeq/g-humin. The metagenomic sequencing of 16S rRNA genes showed the predominance of Firmicutes (71.8 ± 2.5%) in the consortium, and Lachnospiraceae and Ruminococcaceae were considered as the CO 2 -reducing acetogens in the consortium. Thus, microbial fixation of CO 2 using humin introduces new insight to the holistic approach for sustainable treatment of contaminants in environment.

scholarly journals Diversity and Ubiquity of Bacteria Capable of Utilizing Humic Substances as Electron Donors for Anaerobic Respiration

2002 ◽  
Vol 68 (5) ◽  
pp. 2445-2452 ◽  
Author(s):  
John D. Coates ◽  
Kimberly A. Cole ◽  
Romy Chakraborty ◽  
Susan M. O'Connor ◽  
Laurie A. Achenbach
Keyword(s):  
Energy Source ◽  
Carbon Source ◽  
Humic Substances ◽  
Electron Donor ◽  
Electron Acceptor ◽  
Electron Donors ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Couple Growth ◽  
Probable Number

ABSTRACT Previous studies have demonstrated that reduced humic substances (HS) can be reoxidized by anaerobic bacteria such as Geobacter, Geothrix, and Wolinella species with a suitable electron acceptor; however, little is known of the importance of this metabolism in the environment. Recently we investigated this metabolism in a diversity of environments including marine and aquatic sediments, forest soils, and drainage ditch soils. Most-probable-number enumeration studies were performed using 2,6-anthrahydroquinone disulfonate (AHDS), an analog for reduced HS, as the electron donor with nitrate as the electron acceptor. Anaerobic organisms capable of utilizing reduced HS as an electron donor were found in all environments tested and ranged from a low of 2.31 × 101 in aquifer sediments to a high of 9.33 × 106 in lake sediments. As part of this study we isolated six novel organisms capable of anaerobic AHDS oxidation. All of the isolates coupled the oxidation of AHDS to the reduction of nitrate with acetate (0.1 mM) as the carbon source. In the absence of cells, no AHDS oxidation was apparent, and in the absence of AHDS, no cell density increase was observed. Generally, nitrate was reduced to N2. Analysis of the AHDS and its oxidized form, 2,6-anthraquinone disulfonate (AQDS), in the medium during growth revealed that the anthraquinone was not being biodegraded as a carbon source and was simply being oxidized as an energy source. Determination of the AHDS oxidized and nitrate reduced accounted for 109% of the theoretical electron transfer. In addition to AHDS, all of these isolates could also couple the oxidation of reduced humic substances to the reduction of nitrate. No HS oxidation occurred in the absence of cells and in the absence of a suitable electron acceptor, demonstrating that these organisms were capable of utilizing natural HS as an energy source and that AHDS serves as a suitable analog for studying this metabolism. Alternative electron donors included simple volatile fatty acids such as propionate, butyrate, and valerate as well as simple organic acids such as lactate and pyruvate. Analysis of the complete sequences of the 16S rRNA genes revealed that the isolates were not closely related to each other and were phylogenetically diverse, with members in the alpha, beta, gamma, and delta subdivisions of the Proteobacteria. Most of the isolates were closely related to known genera not previously recognized for their ability to couple growth to HS oxidation, while one of the isolates represented a new genus in the delta subclass of the Proteobacteria. The results presented here demonstrate that microbial oxidation of HS is a ubiquitous metabolism in the environment. This study represents the first description of HS-oxidizing isolates and demonstrates that microorganisms capable of HS oxidation are phylogenetically diverse.

scholarly journals Microdiversity and phylogeographic diversification of bacterioplankton in pelagic freshwater systems revealed through long-read amplicon sequencing

Microbiome ◽  
2021 ◽  
Vol 9 (1) ◽  
Author(s):  
Yusuke Okazaki ◽  
Shohei Fujinaga ◽  
Michaela M. Salcher ◽  
Cristiana Callieri ◽  
Atsushi Tanaka ◽  
...  
Keyword(s):  
16S Rrna ◽  
Regional Scale ◽  
Scale Up ◽  
Amplicon Sequencing ◽  
Freshwater Ecosystems ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Metagenomic Sequencing ◽  
Long Read

Abstract Background Freshwater ecosystems are inhabited by members of cosmopolitan bacterioplankton lineages despite the disconnected nature of these habitats. The lineages are delineated based on > 97% 16S rRNA gene sequence similarity, but their intra-lineage microdiversity and phylogeography, which are key to understanding the eco-evolutional processes behind their ubiquity, remain unresolved. Here, we applied long-read amplicon sequencing targeting nearly full-length 16S rRNA genes and the adjacent ribosomal internal transcribed spacer sequences to reveal the intra-lineage diversities of pelagic bacterioplankton assemblages in 11 deep freshwater lakes in Japan and Europe. Results Our single nucleotide-resolved analysis, which was validated using shotgun metagenomic sequencing, uncovered 7–101 amplicon sequence variants for each of the 11 predominant bacterial lineages and demonstrated sympatric, allopatric, and temporal microdiversities that could not be resolved through conventional approaches. Clusters of samples with similar intra-lineage population compositions were identified, which consistently supported genetic isolation between Japan and Europe. At a regional scale (up to hundreds of kilometers), dispersal between lakes was unlikely to be a limiting factor, and environmental factors or genetic drift were potential determinants of population composition. The extent of microdiversification varied among lineages, suggesting that highly diversified lineages (e.g., Iluma-A2 and acI-A1) achieve their ubiquity by containing a consortium of genotypes specific to each habitat, while less diversified lineages (e.g., CL500-11) may be ubiquitous due to a small number of widespread genotypes. The lowest extent of intra-lineage diversification was observed among the dominant hypolimnion-specific lineage (CL500-11), suggesting that their dispersal among lakes is not limited despite the hypolimnion being a more isolated habitat than the epilimnion. Conclusions Our novel approach complemented the limited resolution of short-read amplicon sequencing and limited sensitivity of the metagenome assembly-based approach, and highlighted the complex ecological processes underlying the ubiquity of freshwater bacterioplankton lineages. To fully exploit the performance of the method, its relatively low read throughput is the major bottleneck to be overcome in the future.

Perchlorate removal using two component biodegradable carriers in particle-fixed biofilm reactor

2014 ◽  
Vol 49 (3) ◽  
pp. 234-244
Author(s):  
Fang He ◽  
Fusheng Li ◽  
Haihong Zhou ◽  
Lingling Niu ◽  
Liguo Wang
Keyword(s):  
Carbon Source ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Polymer Particle ◽  
Biofilm Reactor ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Biofilm Reactors ◽  
Gradient Gel Electrophoresis ◽  
Perchlorate Removal ◽  
Fixed Biofilm

In this research, biocompounds designed out of two polymers having different degradability was investigated for use as the sole carbon source and biofilm carrier to remove perchlorate in particle-fixed biofilm reactors. Both laboratory batch and column experiments were conducted with perchlorate contaminated groundwater. Batch experiments demonstrated clearly that ClO4– was removed from the aqueous phase readily and the degradation rate constants (k) changed in the range of 0.23–0.37 mg/L h as ClO4– concentration increased from 2 to 8 mg/L. Simultaneous perchlorate and nitrate degradation occurred in the polymer bioreactor. Effluent concentrations of perchlorate varied positively with temperature and fitted the Arrhenius equation expression as k=k20•100.0316(t–20) over the range of 13–30 °C. No perchlorate was detected in the effluent of polymer columns after 20 days’ startup. Complete perchlorate removal was observed at a hydraulic loading rate doubled to 1.8 mL/min. Images prove the concept of the pore and filament structure within the biocompounds, which provide both a heterotrophic biofilm and carbon source. Denaturing gradient gel electrophoresis analysis and partial sequencing of 16S rRNA genes indicated that formerly reported perchlorate-reducing bacteria were present in the polymer particle-fixed biofilm reactors.

Abundance, diversity and mobility potential of antibiotic resistance genes in pristine Tibetan Plateau soil as revealed by soil metagenomics

2020 ◽  
Vol 96 (10) ◽  
Author(s):  
Bo Li ◽  
Zeng Chen ◽  
Fan Zhang ◽  
Yongqin Liu ◽  
Tao Yan
Keyword(s):  
Antibiotic Resistance ◽  
Microbial Diversity ◽  
Resistance Genes ◽  
Antibiotic Resistance Genes ◽  
Soil Samples ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Metagenomic Sequencing ◽  
Clinical Issue ◽  
Abundance And Diversity

ABSTRACT Widespread occurrence of antibiotic resistance genes (ARGs) has become an important clinical issue. Studying ARGs in pristine soil environments can help to better understand the intrinsic soil resistome. In this study, 10 soil samples were collected from a high elevation and relatively pristine Tibetan area, and metagenomic sequencing and bioinformatic analyses were conducted to investigate the microbial diversity, the abundance and diversity of ARGs and the mobility potential of ARGs as indicated by different mobile genetic elements (MGEs). A total of 48 ARG types with a relative abundance of 0.05–0.28 copies of ARG/copy of 16S rRNA genes were detected in Tibetan soil samples. The observed ARGs were mainly associated with antibiotics that included glycopeptide and rifamycin; the most abundant ARGs were vanRO and vanSO. Low abundance of MGEs and potentially plasmid-related ARGs indicated a low horizontal gene transfer risk of ARGs in the pristine soil. Pearson correlation and redundancy analyses showed that temperature and total organic carbon were the major environmental factors controlling both microbial diversity and ARG abundance and diversity.

scholarly journals BIOAUGMENTATION OF ACRYLAMIDE-DEGRADING BACTERIA IN THE MICROBIOTA OF RIVER SLUDGE

2021 ◽  
Vol 26 (3) ◽  
pp. 56-65
Author(s):  
Yu. G. Maksimova ◽  
◽  
G. V. Ovechkina ◽  
A. Yu. Maksimov ◽  
◽  
...  
Keyword(s):  
Phylogenetic Diversity ◽  
Toxic Substance ◽  
Alcaligenes Faecalis ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Small Rivers ◽  
Metagenomic Sequencing ◽  
Degrading Bacteria ◽  
Acinetobacter Guillouiae

Introduction. Bioaugmentation is an in situ bioremediation approach, which implies the introduction of a population of microorganisms with certain biodegrading abilities. Acrylamide is a biodegradable toxic substance. Our goal was to assess the survival of allochthonous bacterial cultures Alcaligenes faecalis 2 and Acinetobacter guillouiae 11h when introduced into river sludge and the efficiency of acrylamide decomposition by sludge with introduced amidase-containing bacteria. Methods. The microbiota of sludge from small rivers of Perm Territory was inoculated with the biomass of strains A. faecalis 2 and A. guillouiae 11h, which have amidase activity. In a laboratory experiment, we studied the survival of these bacteria as well as the biodegrading ability of the microbiota in relation to acrylamide after 3 and 6 months of incubation at 5 and 25°C. The transformation of acrylamide was assessed by HPLC, the biodiversity of river sludge was assessed by the method of metagenomic sequencing of 16S rRNA genes. Results. Incubation of sludge at 25°C for 3–6 months deteriorates the biodegrading abilities of the microbiota in relation to acrylamide, and the transformation of this pollutant occurs only during the augmentation of the biomass of amidase-containing bacteria, with acinetobacteria having an advantage over bacteria of Alcaligenes sp. Upon incubation of sludge at 25°C, the phylogenetic diversity increases, and the proportion of representatives of the phyla Actinobacteria, Chloroflexi, Ignavibacteriae, Candidatus Saccharibacteria, Acidobacteria increases as well, while the phylum Proteobacteria accounts for most of the bacterial biota in all samples, and the phylum Firmicutes accounts for 10–30%. The presence of representatives of Alcaligenes sp. and Acinetobacter sp. was confirmed in the microbiota of bioaugmented sludge after 6 months of incubation at 25°C. When incubated at 5°C, the microbiota of native sludge is capable of degrading acrylamide, but at a rate several times lower than during bioaugmentation. After incubation of Danilikha River sludge with the introduced biomass of strains A. guillouiae 11h and A. faecalis 2 at 5°C for 6 months, the complete transformation of acrylamide was observed in 4 and 20 days, respectively, with native sludge — in 35 days.

scholarly journals Vertical Distribution of Methanogens in the Anoxic Sediment of Rotsee (Switzerland)

1999 ◽  
Vol 65 (6) ◽  
pp. 2402-2408 ◽  
Author(s):  
K. Zepp Falz ◽  
C. Holliger ◽  
R. Großkopf ◽  
W. Liesack ◽  
A. N. Nozhevnikova ◽  
...  
Keyword(s):  
Methane Production ◽  
Dry Weight ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Sediment Layer ◽  
Anoxic Sediment ◽  
Dapi Staining ◽  
Methanogenic Activity ◽  
Hydrogenotrophic Methanogenesis ◽  
Chemical Profiles

ABSTRACT Anoxic sediments from Rotsee (Switzerland) were analyzed for the presence and diversity of methanogens by using molecular tools and for methanogenic activity by using radiotracer techniques, in addition to the measurement of chemical profiles. After PCR-assisted sequence retrieval of the 16S rRNA genes (16S rDNA) from the anoxic sediment of Rotsee, cloning, and sequencing, a phylogenetic analysis identified two clusters of sequences and four separated clones. The sequences in cluster 1 grouped with those of Methanosaeta spp., whereas the sequences in cluster 2 comprised the methanogenic endosymbiont ofPlagiopyla nasuta. Discriminative oligonucleotide probes were constructed against both clusters and two of the separated clones. These probes were used subsequently for the analysis of indigenous methanogens in a core of the sediment, in addition to domain-specific probes against members of the domains Bacteria andArchaea and the fluorescent stain 4′,6-diamidino-2-phenylindole (DAPI), by fluorescent in situ hybridization. After DAPI staining, the highest microbial density was obtained in the upper sediment layer; this density decreased with depth from (1.01 ± 0.25) × 1010 to (2.62 ± 0.58) × 1010 cells per g of sediment (dry weight). This zone corresponded to that of highest metabolic activity, as indicated by the ammonia, alkalinity, and pH profiles, whereas the methane profile was constant. Probes Eub338 and Arch915 detected on average 16 and 6% of the DAPI-stained cells as members of the domains Bacteriaand Archaea, respectively. Probe Rotcl1 identified on average 4% of the DAPI-stained cells as Methanosaeta spp., which were present throughout the whole core. In contrast, probe Rotcl2 identified only 0.7% of the DAPI-stained cells as relatives of the methanogenic endosymbiont of P. nasuta, which was present exclusively in the upper 2 cm of the sediment. Probes Rotp13 and Rotp17 did not detect any cells. The spatial distribution of the two methanogenic populations corresponded well to the methane production rates determined by incubation with either [14C]acetate or [14C]bicarbonate. Methanogenesis from acetate accounted for almost all of the total methane production, which concurs with the predominance of acetoclastic Methanosaeta spp. that represented on average 91% of the archaeal population. Significant hydrogenotrophic methanogenesis was found only in the organically enriched upper 2 cm of the sediment, where the probably hydrogenotrophic relatives of the methanogenic endosymbiont of P. nasuta, accounting on average for 7% of the archaeal population, were also detected.

The difference of the gut microbiota of gastric cancer in relation to Helicobacter pylori negativity and positivity.

2019 ◽  
Vol 37 (4_suppl) ◽  
pp. 10-10
Author(s):  
Mijin Seol ◽  
Yu Ra Lee ◽  
Kyung Mi Kim ◽  
Cheol Min Shin ◽  
Hyuk Yoon ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Helicobacter Pylori ◽  
Relative Abundance ◽  
Fecal Sample ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Bacillus Species ◽  
Sequencing Analysis ◽  
Metagenomic Sequencing ◽  
Microbial Composition

10 Background: Helicobacter pylori (HP) is a major risk factor for gastric cancer, however, only 1-2% of HP(+) people develop adenocarcinoma. In this study, we have compared the intestinal microbiota composition related to HP status among gastric cancer patient using 16SrRNA gene-based metagenomic sequencing analysis and culture-based method. Methods: Stool samples were collected from 18 gastric cancer patients. 16S rRNA genes were sequenced on the Illumina Miseq platform and further analyzed to evaluate the gut bacterial community. The bacteria strains of fecal sample were isolated in aerobic and anaerobic condition. Results: Metagenomics analysis of fecal sample showed four major phyla; Firmicutes, Bacteroidetes, Proteobacteria and Actinobacteria were dominant. Firmicutes were the most dominant phylum. Within this phylum, the relative abundance of Clostridiales including Ruminococcus was higher in the HP(-) group, whereas Lactobacillales including streptococcus was higher in HP(+) group. In addition the relative abundance of Bacteroidetes in HP(-) group and Actinobacteria (especially, genus Bifidobacterium) in HP(+) group was observed highly. In the bacterial culture-based approach, bacteria strains belonged to Clostridiales such as Clostridium perfringens, Ruminococcus feacis, Blautia sp., Coprococcus comes were isolated in HP(-) sample. In HP(+) sample, Klebsiella, Bacteroides, Bifidobacterium were isolated. Bacillus species, Escherichia/Shigella was enriched regardless of HP exist. Streptococcus was not cultivated in HP(+) group, but isolated in HP(-) group in contrast with metagenome data. Conclusions: We found the intestinal bacterial diversity in the HP(+) group was lower than those in the HP(-) and the microbial composition was different between HP(+) and HP(-). Metagenome analysis showed the order Clostridiales of the phylum Firmicutes were enriched in the HP(-) group while the order Lactobacillales (specially, Streptococcus) were enriched in the HP(+) group. Compared to isolates between two groups, bacteria species belonged to the order Clostridiales such as Clostridium, Ruminococcus , Blautia , Coprococcus were cultivated particularly in HP(-) sample.

Sputum Microbiota in Coal Workers Diagnosed With Pneumoconiosis as Revealed by 16S Metagenomic Sequencing

2020 ◽  
Author(s):  
Vladimir Druzhinin ◽  
Liudmila Matskova ◽  
Pavel Demenkov ◽  
Elizaveta Baranova ◽  
Valentin Volobaev ◽  
...  
Keyword(s):  
Inflammatory Responses ◽  
Bacterial Species ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Occupational Activity ◽  
Metagenomic Sequencing ◽  
Coal Miners ◽  
Active Coal ◽  
Coal Workers ◽  
Generation Sequencing

Abstract Objectives: The microbiome of sputum from former and active coal miners diagnosed with coal worker’s pneumoconiosis (CWP) as compared to healthy controls Methods: Next Generation Sequencing of bacterial 16S rRNA genes obtained from the sputum of CWP subjects.Results: Differences were detected between the sputum microbiomes from the healthy and CWP subjects. We noted a significant decrease in Bacteroidetes and an increase in the level of Proteobacteria.Conclusions: The microbiomes found in sputum from CWP subjects are enriched in bacterial species previously reported to induce pro-inflammatory responses. The profile of the microbiomes correlated mainly to the occupational activity and not to the age of the coal miners.

Thermal pretreatment of the solid fraction of manure: impact on the biogas reactor performance and microbial community

2006 ◽  
Vol 53 (8) ◽  
pp. 59-67 ◽  
Author(s):  
Z. Mladenovska ◽  
H. Hartmann ◽  
T. Kvist ◽  
M. Sales-Cruz ◽  
R. Gani ◽  
...  
Keyword(s):  
Microbial Community ◽  
Solid Fraction ◽  
Swine Manure ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Significant Activity ◽  
Reactor Performance ◽  
Methanogenic Activity ◽  
Stirred Tank Reactors ◽  
Biogas Reactor

Application of thermal treatment at 100–140 °C as a pretreatment method prior to anaerobic digestion of a mixture of cattle and swine manure was investigated. In a batch test, biogasification of manure with thermally pretreated solid fraction proceeded faster and resulted in the increase of methane yield. The performances of two thermophilic continuously stirred tank reactors (CSTR) treating manure with solid fraction pretreated for 40 minutes at 140 °C and non-treated manure were compared. The digester fed with the thermally pretreated manure had a higher methane productivity and an improved removal of the volatile solids (VS). The properties of microbial communities of both reactors were analysed. The specific methanogenic activity (SMA) test showed that both biomasses had significant activity towards hydrogen and formate, while the activity with the VFA – acetate, propionate and butyrate – was low. The kinetic parameters of the VFA conversion revealed a reduced affinity of the microbial community from the CSTR fed with thermally pre-treated manure for acetate, propionate and butyrate. The bacterial and archaeal populations identified by t-RLFP analysis of 16S rRNA genes were found to be identical in both systems. However, a change in the abundance of the species present was detected.

scholarly journals Ultradeep Microbial Communities at 4.4 km within Crystalline Bedrock: Implications for Habitability in a Planetary Context

Life ◽  
2020 ◽  
Vol 10 (1) ◽  
pp. 2 ◽  
Author(s):  
Lotta Purkamo ◽  
Riikka Kietäväinen ◽  
Maija Nuppunen-Puputti ◽  
Malin Bomberg ◽  
Claire Cousins
Keyword(s):  
Microbial Communities ◽  
Marker Gene ◽  
Its Region ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Marker Genes ◽  
Metagenomic Sequencing ◽  
Deep Biosphere ◽  
Crystalline Bedrock ◽  
Geothermal Drilling

The deep bedrock surroundings are an analog for extraterrestrial habitats for life. In this study, we investigated microbial life within anoxic ultradeep boreholes in Precambrian bedrock, including the adaptation to environmental conditions and lifestyle of these organisms. Samples were collected from Pyhäsalmi mine environment in central Finland and from geothermal drilling wells in Otaniemi, Espoo, in southern Finland. Microbial communities inhabiting the up to 4.4 km deep bedrock were characterized with phylogenetic marker gene (16S rRNA genes and fungal ITS region) amplicon and DNA and cDNA metagenomic sequencing. Functional marker genes (dsrB, mcrA, narG) were quantified with qPCR. Results showed that although crystalline bedrock provides very limited substrates for life, the microbial communities are diverse. Gammaproteobacterial phylotypes were most dominant in both studied sites. Alkanindiges -affiliating OTU was dominating in Pyhäsalmi fluids, while different depths of Otaniemi samples were dominated by Pseudomonas. One of the most common OTUs detected from Otaniemi could only be classified to phylum level, highlighting the uncharacterized nature of the deep biosphere in bedrock. Chemoheterotrophy, fermentation and nitrogen cycling are potentially significant metabolisms in these ultradeep environments. To conclude, this study provides information on microbial ecology of low biomass, carbon-depleted and energy-deprived deep subsurface environment. This information is useful in the prospect of finding life in other planetary bodies.

Sign in / Sign up

Export Citation Format

Share Document