Search Results (64)

A novel actinobacterial strain, designated E54^T, was isolated from a hyper-arid desert soil sample collected from the Kumtagh Desert in Dunhuang, Gansu Province, China. Phylogenetic analysis based on 16S rRNA gene sequences placed strain E54^T within the genus Lentzea, showing highest similarity to Lentzea waywayandensis DSM 44232^T (98.9%) and Lentzea flava NBRC 15743^T (98.5%). However, whole-genome comparisons revealed that the average nucleotide identity (ANI) and digital DNA–DNA hybridization (dDDH) values between E54^T and these related strains were below the thresholds for species delineation. Strain E54^T exhibited typical morphological characteristics of the genus Lentzea, forming a branched substrate. It grew optimally at 28–30 °C, pH 7.0–9.0, and tolerated up to 10% NaCl. The cell wall contained meso-diaminopimelic acid, the predominant menaquinone was MK-9(H₄), and major fatty acids included iso-C_16:0. The polar lipid profile comprised diphosphatidyl glycerol, phosphatidyl ethanolamine, phosphatidyl inositol, hydroxyphosphatidyl ethanolamine, and an unidentified lipid. The characteristic amino acid type of the cell wall was meso-DAP. Whole-cell hydrolysis experiments revealed the characteristic cell wall sugar fractions: ribose and galactose. The genome of strain E54^T is approximately 8.0 Mb with a DNA G+C content of 69.38 mol%. Genome mining revealed 39 biosynthetic gene clusters (BGCs), including non-ribosomal peptide synthetases (NRPS), polyketide synthases (PKS), terpenes, and siderophores. Comparative antiSMASH-based genome analysis across 38 Lentzea strains further demonstrated the genus’ remarkable biosynthetic diversity. NRPS and type I PKS (T1PKS) were the most prevalent BGC types, indicating a capacity to synthesize structurally complex and pharmacologically relevant metabolites. Together, these findings underscore the untapped biosynthetic potential of the genus Lentzea and support the proposal of strain E54^T as a novel species. The strain E54^T (=JCM 34936^T = GDMCC 4.216^T) should represent a novel species, for which the name Lentzea xerophila sp. nov. is proposed. Full article

ES5-4^T, a Gram-positive, motile, aerobic, and rod-shaped strain, was isolated from the rhizosphere of Galinsoga parviflora growing in the selenium-rich ore area of Enshi, Hubei Province, China. This strain can grow at pH levels of 5.0–10.0 and temperatures of 4–42 °C, with optimal growth at pH 7.0 and 28 °C. It was found to resist NaCl up to 5% (w/v), with an optimal growth condition of 0.5–1.0%. The strain exhibited tolerance to selenite (Se⁴⁺) concentrations up to 5000 mg/L. The major fatty acids of the ES5-4^T strain were anteiso-C_15:0 (46.5%) and C_16:0 (21.7%), its predominant respiratory quinone was MK-7, and its polar lipids included diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and an unidentified phospholipid (PL). The presence of the 16S rRNA gene sequence implies that ES5-4^T belongs to a member of the genus Paenibacillus, with the highest sequence similarity of 98.4% to Paenibacillus pabuli NBRC 13638^T. The bac120 tree also confirmed that the strain is within the genus Paenibacillus. The average nucleotide identity (ANI) and digital DNA–DNA hybridization (dDDH) values between ES5-4^T and closely related members of the genus Paenibacillus were all below the cutoff levels of 95–96% and 70%, respectively. Based on a polyphasic approach, including phenotypic, chemotaxonomic, and phylogenetic analyses, the ES5-4^T strain is proposed as a novel species of the genus Paenibacillus, for which the name Paenibacillus hubeiensis sp. nov. is proposed. This type strain is designated as ES5-4^T (=GDMCC 1.3540^T = KCTC 43478^T). Full article

Interest in the production of bioethanol from inedible biomass is growing worldwide because of its sustainable supply and lack of competition with food supplies. Candida krusei (also known as Pichia kudriavzevii or Issatchenkia orientalis) is one of the most suitable thermotolerant yeasts used in the simultaneous saccharification and fermentation process for bioethanol production. In the production of bioethanol from lignocellulosic biomass as a feedstock, various environmental conditions occur, and the stress tolerance capacity of C. krusei, especially its low pH and tolerance to inhibitors, limits its practical application. In this study, to select a suitable second-generation bioethanol-producing strain, the tolerance capacity of five available C. krusei strains (NBRC0584, NBRC0841, NBRC1162, NBRC1395 and NBRC1664) was characterized. Spot assay and growth experiment results showed that among the five C. krusei strains, C. krusei NBRC1664 showed superior tolerance capacity for low pH and inhibitors. Furthermore, this strain efficiently produced ethanol from glucose under low pH conditions with or without sulfate. A comparative analysis of the draft genome sequences suggested that Opy2, Sln1 and Cdc24 in the HOG pathway are conserved only in C. krusei NBRC1664, which may contribute to its superior tolerance to low pH levels. Moreover, amino acid sequence alignment showed that aldehyde dehydrogenase family proteins, which catalyze the degradation of cyclic aldehydes, are commonly conserved in C. krusei. In addition, the increased transcription levels in C. krusei NBRC1664 could play a role in its higher tolerance to inhibitors. These results suggest that C. krusei NBRC1664 is a more suitable strain for application in industrial processes for second-generation bioethanol production. Full article

Hadal zones account for the deepest 45% of oceanic depth range and play an important role in ocean biogeochemical cycles. As the least-explored aquatic habitat on earth, further investigation is still required to fully elucidate the microbial taxonomy, ecological significance, metabolic diversity, and adaptation in hadal environments. In this study, a novel strain Lsc_1132^T was isolated from sediment of the Mariana Trench at 10,954 m in depth. Strain Lsc_1132^T contains heterogenous 16S rRNA genes, exhibiting the highest sequence similarities to the type strains of Neobacillus drentensis LMG 21831^T, Neobacillus dielmonensis, Neobacillus drentensis NBRC 102427^T, Neobacillus rhizosphaerae, and Neobacillus soli NBRC 102451^T, with a range of 98.60–99.10% identity. The highest average nucleotide identity (ANI), the highest digital DNA-DNA hybridization (DDH) values, and the average amino acid identity (AAI) with Neobacillus sp. PS3-40 reached 73.5%, 21.4%, and 75.54%, respectively. The major cellular fatty acids of strain Lsc_1132^T included iso-C_15:0, Summed Feature 3 (C_16:1ω6c and/or C_16:1ω7c), iso-C_17:0, anteiso-C_15:0, and iso-C_17:1ω5c. The respiratory quinone of strains Lsc_1132^T was MK-7. The G + C content of the genomic DNA was 40.9%. Based on the GTDB taxonomy and phenotypic data, strain Lsc_1132^T could represent a novel species of a novel genus, proposed as Aliineobacillus hadale gen. nov. sp. nov. (type strain Lsc_1132^T = MCCC 1K09620^T). Metabolically, strain Lsc_1132^T demonstrates a robust carbohydrate metabolism with many strain-specific sugar transporters. It also has a remarkable capacity for metabolizing amino acids and carboxylic acids. Genomic analysis reveals a streamlined genome in the organism, characterized by a significant loss of orthologous genes, including those involved in cytochrome c synthesis, aromatic compound degradation, and polyhydroxybutyrate (PHB) synthesis, which suggests its adaptation to low oxygen levels and oligotrophic conditions through alternative metabolic pathways. In addition, the reduced number of paralogous genes in strain Lsc_1132^T, together with its high protein-coding gene density, may further contribute to streamlining its genome and enhancing its genomic efficiency. This research expands our knowledge of hadal microorganisms and their metabolic strategies for surviving in extreme deep-sea environments. Full article

Enterococcus faecalis (E. faecalis) has been associated with the specific production of l-(+)-lactic acid. However, in this study, d-(−)-lactic acid production by E. faecalis was observed under specific pH conditions. E. faecalis PR31 exhibited a significant amount of d-(−)-lactic acid under a stirring culture in MRS broth at pH 4.5, 5.8, and 6.0, and the contents of d-(−)-lactic acid were 45.1, 35.9, and 36.2%, respectively. When the cell suspension prepared at a pH of 6.0 was reacted with l-(+)- or d-(−)-lactic acid, d-(−)- or l-(+)-lactic acid was produced, respectively, in a time- and dose-dependent manner. Therefore, this phenomenon of d-(−)-lactic acid production in PR31 was suggested to be due to the activation of the larA gene encoding lactate racemase that is present in PR31. However, even in the E. faecalis-type strain NBRC 100480, which contains neither larA nor vanH, encoding d-(−)-lactate dehydrogenase VanH, d-(−)-lactic acid was also produced at specific pH values. Therefore, the production of d-(−)-lactic acid in NBRC 100480 was thought to occur not via the activation of larA. The biological significance of d-(−)-lactic acid production in E. faecalis depending on the pH and the detailed underlying mechanism, including whether it is the same in PR31 and NBRC 100480, remain to be elucidated in future studies. Full article

The increase in infections derived from biofilms from Staphylococcal spp. prompted us to develop novel strategies to inhibit biofilm development. Nanoscale protrusion structures (nanopillars) observed on the wings of dragonflies and cicadas have recently gained notable attention owing to their physical, antimicrobial, and bactericidal properties. Thus, they are not only expected to reduce the damage caused by chemical antimicrobial agents to human health and the environment, but also to serve as a potential countermeasure against the emergence of antimicrobial-resistant bacteria (ARB). In this study, we evaluated the anti-biofilm effects of cyclo-olefin polymer (COP) nanopillars by changing the wettability of surfaces ranging in height from 100 to 500 nm against Staphylococcus spp., such as Staphylococcus aureus NBRC 100910 (MSSA), Staphylococcus aureus JCM 8702 methicillin-resistant S. aureus (MRSA), and Staphylococcus epidermidis ATCC 35984. The results clearly show that the fabricated nanopillar structures exhibited particularly strong biofilm inhibition against MRSA, with inhibition rates ranging from 51.2% to 62.5%. For MSSA, anti-biofilm effects were observed only at nanopillar heights of 100–300 nm, with relatively low hydrophobicity, with inhibition rates ranging from 23.9% to 40.8%. Conversely, no significant anti-biofilm effect was observed for S. epidermidis in any of the nanopillar structures. These findings suggest that the anti-biofilm properties of nanopillars vary among bacteria of the same species. In other words, by adjusting the height of the nanopillars, selective anti-biofilm effects against specific bacterial strains can be achieved. Full article

Microorganisms from poorly explored environments are promising sources for the development of novel drugs. In our continuous efforts to screen for mangrove actinomycetes that produce metabolites with potential pharmaceutical applications, Streptomyces sp. Y009 was isolated from mangrove sediments in Guangxi, China. The phenotypic, physiological, biochemical, and phylogenetic characteristics of this strain were investigated. Analysis of phylogenetic and 16S rRNA gene sequences showed that it had the highest sequence similarity to Streptomyces thermolilacinus NBRC 14274 (98.95%). Further, the Y009 extract exhibited antioxidant activity, as indicated by DPPH and superoxide dismutase assays. The extract showed broad-spectrum and potent anticancer potential against six human cancer cell lines, with IC₅₀ values ranging from 5.61 to 72.15 μg/mL. Furthermore, the selectivity index (SI) demonstrated that the Y009 extract exhibited less toxicity toward normal cell lines in comparison to the lung cancer cell line (A549) and hepatoma cell line (HepG2). GC–MS analysis revealed that the extract contained some biologically important secondary metabolites, mainly cyclic dipeptides and esters, which might be responsible for the antioxidant and anticancer properties. 3-Isobutylhexahydropyrrolo[1,2-a]pyrazine-1,4-dione (28.32%) was the major chemical compound available in the extract. The effect on cancer cells was then confirmed using nuclear staining and in silico docking. This study suggests that further exploration of the bioactive compounds of the newly isolated strain may be a promising approach for the development of novel chemopreventive drugs. Full article

The aim of this study was to examine the biological activity and probiotic properties of lactic acid bacteria (LAB) isolated from sweet potato stalk kimchi (SPK). Various LAB and Bacillus spp. are active in the early stages of the fermentation of kimchi made from sweet potato stalk. Four strains of LAB were identified, including SPK2 (Levilactobacillus brevis ATCC 14869), SPK3 (Latilactobacillus sakei NBRC 15893), SPK8 and SPK9 (Leuconostoc mesenteroides subsp. dextranicum NCFB 529). SPK2, SPK3, SPK8, and SPK9 showed 64.64–94.23% bile acid resistance and 78.66–82.61% pH resistance. We identified over 10⁶ CFU/mL after heat treatment at 75 °C. Four strains showed high antimicrobial activity to Escherichia coli and Salmonella Typhimurium with a clear zone of >11 mm. SPK2 had the highest antioxidative potentials, higher than the other three bacteria, with 44.96 μg of gallic acid equivalent/mg and 63.57% DPPH scavenging activity. These results demonstrate that the four strains isolated from sweet potato kimchi stalk show potential as probiotics with excellent antibacterial effects and may be useful in developing health-promoting products. Full article

Pear black spot, caused by A. gaisen during fruit growth, is a disease that significantly reduces pear yield. Biological control using antagonistic microorganisms is regarded as a viable alternative to chemical agents. The discovery of TRM76147, a novel species of Streptomyces isolated from the Taklamakan Desert, has demonstrated promising potential in addressing this issue. This study was conducted to determine the potential of crude extract of Streptomyces sp. nov., strain TRM76147, for control of A. gaisen. TRM76147 is closely related to Streptomyces griseoviridis NBRC 12874^T, exhibiting an average nucleotide identity (ANI) value of 82.13%. Combined with the polyphasic taxonomic identification, this suggests that TRM76147 is a potentially new species. Through analyses using BigSCAPE and antiSMASH, it was determined that the TRM76147 genome contains 19 gene clusters. The ethyl acetate extract of this strain demonstrates antifungal activity, with the active substance remaining stable at temperatures up to 70 °C, achieving an activity level of 16.23 ± 0.22 mm. Furthermore, the crude extract maintains its antifungal efficacy across a pH range of 2 to 12. Notably, the antifungal diameter was recorded at 16.53 ± 0.12 mm following 80 min of UV irradiation. Under different treatment conditions, TRM76147 fermentation crude extract caused A. gaisen spore crumpling and spore number reduction. In addition, this study also found that the TRM76147 fermentation broth could control the production of pear black spot disease, which initially revealed the inhibition mechanism. The abundant actinomycete resources in this study have good application and development value in the discovery of new species and the study of bioactive substances and biological control. Full article

Butirosins are naturally occurring aminoglycoside (AG) antibiotics featuring a 4,5-disubstituted 2-deoxystreptamine (2-DOS) with a (2S)-4-amino-2-hydroxybutyrate (AHBA) side chain. This side chain has been shown to confer resistance against AG-modifying enzymes, leading to ongoing studies on the butirosin biosynthetic pathway and the corresponding enzymes. Butirosin is produced by Niallia (formerly Bacillus) circulans and Bacillus vitellinus, with most research focused on the first strain. To date, no whole-genome analysis has been performed on B. vitellinus. In this study, we sequenced the complete genome of B. vitellinus NBRC 13296 and performed a comparative analysis of different butirosin biosyntheric gene clusters (BGCs), including those from N. circulans. The complete genome of B. vitellinus NBRC 13296 comprises a 6,331,192-base circular chromosome with GC content of 52.68%. The annotation revealed the presence of 5605 CDSs, 70 tRNA genes, 30 rRNA genes, and 3 ncRNA genes in NBRC 13296. The highest dDDH and ANI values between NBRC 13296 and the most closely related type strain, Paenibacillus chitinolyticus KCCM 41,400, were 97.8% and 98.66%, respectively. Based on these genome-based comparative analyses, we propose reclassifying B. vitellinus NBRC 13296 as P. chitinolyticus. Genome mining revealed 18 gene clusters encoding the biosynthesis of diverse secondary metabolites in the genome of B. vitellinus NBRC 13296, indicating the enormous biosynthetic potential of this strain. The predicted structural diversity of the secondary metabolites includes aminoglycosides, PKS, NRPS, PKS–NRPS hybrids, metallophores, phosphonates, terpenes, β-lactones, and RiPP peptides. We then comparatively characterized the butirosin BGCs previously studied in several N. circulans strains. Additionally, the comparative genome analysis revealed complete butirosin BGCs identified from P. chitinolyticus KCCM 41,400, P. chitinolyticus NRRL B-23119, P. chitinolyticus NRRL B-23120, P. chitinolyticus B-14908, P. chitinolyticus YSY-3.1, P. chitinolyticus JMW06, Paenibacillus sp. GbtcB18, Paenibacillus sp. HGH0039, and Paenibacillus sp. MZ04-78.2. Finally, we identified the core region consisting of BtrS, BtrN, BtrM, BtrL, BtrA, BtrB, BtrC, BtrD, BtrD, BtrE, BtrF, BtrG, BtrH, BtrI, BtrI, BtrJ, BtrK, BtrO, BtrP, and BtrV, followed by an upstream region organizing BtrQ, BtrW, BtrX, BtrY, and BtrZ in the same transcriptional direction and sequential genetic arrangement, and a downstream region organizing various proteins based on BtrT, BtrR2, BtrU, and BtrR1. Our study provides insights into the reclassification of B. vitellinus NBRC 13296 to P. chitinolyticus and suggests the need for continued studies on butirosin biosynthesis from an enzymatic perspective. Full article

A Gram-stain-negative, obligately aerobic, non-motile, rod-shaped bacterial strain designated SJW1-29^T was isolated from brackish water samples collected from the Seomjin River, Republic of Korea. The purpose of this study was to characterize strain SJW1-29^T and determine its taxonomic position as a potential new genus within the family Spirosomaceae. The strain grew within the range of 10–30 °C (optimum, 25 °C), pH 5.0–10.0 (optimum, 7.0), and 1–4% NaCl (optimum, 3%). Phylogenetic analysis based on the 16S rRNA gene revealed that strain SJW1-29^T belongs to the family Spirosomaceae and is closely related to Persicitalea jodogahamensis Shu-9-SY12-35C^T (91.3% similarity), Rhabdobacter roseus R491^T (90.6%), and Arundinibacter roseus DMA-K-7a^T (90.0%), while the similarities to strains within the order Cytophagales were lower than 90.0%. The genome is 7.1 Mbp with a G+C content of 50.7 mol%. The use of genome-relatedness indices confirmed that this strain belongs to a new genus. The major polar lipid profile consisted of phosphatidylethanolamine, and MK-7 was the predominant menaquinone. The predominant fatty acids were summed feature 3 (C_16:1 ω7c and/or C_16:1 ω6c), iso-C_15:0, iso-C_17:0 3-OH, and C_16:0, representing more than 80% of the total fatty acids. The phenotypic, chemotaxonomic, genetic, and phylogenetic properties suggest that strain SJW1-29^T represents a novel species within a new genus in the family Spirosomaceae, for which the name Salmonirosea aquatica gen. nov., sp. nov., is proposed. The type strain of Salmonirosea aquatica is SJW1-29^T (=KCTC 72493^T = NBRC 114061^T = FBCC-B16924^T). Full article

The thermotolerant yeast Pichia kudriavzevii (previously known as Issatchenkia orientalis), can produce ethanol from a variety of carbon sources and grows at around 45 °C. Thus, this yeast is considered a useful biocatalyst for producing ethanol from lignocellulose through simultaneous saccharification and fermentation (SSF). SSF has several advantages, such as a simplified manufacturing process, ease of operation and reduced energy input. Using P. kudriavzevii NBRC1279 and NBRC1664, we previously succeeded in producing ethanol through SSF; however, the extent to which inhibitors by-produced from lignocellulose hydrolysis affect the growth and ethanol productivity of the two strains remains to be investigated. In this study, to better understand the inhibitor tolerance capacity of the two strains, spot assay, growth experiment, real-time quantitative PCR (RT-qPCR) analysis and multiple sequence alignment analysis were carried out. When P. kudriavzevii NBRC1279 and NBRC1664, as well as Saccharomyces cerevisiae BY4742 as a control, were cultured on SCD plates containing 17% ethanol, 42 mM furfural, 56 mM 5-hydroxymethylfurfural (HMF) or 10 mM vanillin, only P. kudriavzevii NBRC1664 was able to grow under all conditions. Moreover, the inhibitor tolerance capacity of P. kudriavzevii NBRC1664 was greater than those of other strains using SCD medium containing the same concentrations of various inhibitors. When an RT-qPCR analysis of seven gene sequences from aldehyde dehydrogenase and the aldehyde dehydrogenase family protein (ADHF) was performed using P. kudriavzevii NBRC1664 cultivated in the presence of 56 mM HMF, ADHF1 and ADHF2 were up-regulated in the early logarithmic growth phase. Moreover, a multiple sequence alignment of the amino acid sequences of ADHF1, ADHF2 and the known ADH suggested that ADHF1 and ADHF2 may catalyze the reversible NAD⁺-dependent oxidation of HMF. Our data may be useful for future studies on the metabolic engineering of more useful strains for ethanol production from lignocellulose. Full article

Streptomyces species are attractive sources of secondary metabolites that serve as major sources of antibiotics and other drugs. In this study, genome mining was used to determine the biosynthetic potential of Streptomyces sp. 21So2-11 isolated from Antarctic soil. 16S rRNA gene sequencing revealed that this strain is most closely related to Streptomyces drozdowiczii NBRC 101007^T, with a similarity of 98.02%. Genome comparisons based on average nucleotide identity (ANI) and digital DNA–DNA hybridization (dDDH) showed that strain 21So2-11 represents a novel species of the genus Streptomyces. In addition to a large number of genes related to environmental adaptation and ecological function, a total of 28 putative biosynthetic gene clusters (BGCs) responsible for the biosynthesis of known and/or novel secondary metabolites, including terpenes, lantipeptides, polyketides, nonribosomal peptides, RiPPs and siderophores, were detected in the genome of strain 21So2-11. In addition, a total of 1456 BGCs were predicted to contribute to the biosynthesis of more than 300 secondary metabolites based on the genomes of 47 Streptomyces strains originating from polar regions. The results indicate the potential of Streptomyces sp. 21So2-11 for bioactive secondary metabolite production and are helpful for understanding bacterial adaptability and ecological function in cold terrestrial environments. Full article

Insect farming is gaining attention as a promising area for exploring probiotic bacteria, which can benefit both insect health and various industries. Silkworm farming is a key industry in Thailand; however, challenges such as disease susceptibility and optimising growth require innovative solutions for sustainable practices. Our study addresses this by assessing lactic acid bacteria (LAB) in native Thai silkworm faeces, which accumulate as natural by-products during the rearing process. We conducted biochemical tests, including those for catalase, haemolytic activity, bile salt tolerance, antimicrobial activity, antibiotic susceptibility, and cell surface hydrophobicity, along with taxonomic classification. Out of 102 isolates, eight potential probiotics were selected, with five showing strong probiotic traits like acid and bile salt tolerance and cell surface hydrophobicity, enhancing gut survivability. These isolates also displayed antagonistic activity against pathogens like Staphylococcus aureus, Salmonella typhimurium, Escherichia coli, and Pseudomonas aeruginosa. Safety assessments confirmed their safety, with no haemolytic activity and sensitivity to antibiotics like chloramphenicol and amoxicillin. These LAB isolates (SP04, SP06, SP44, SP64, and SP67), identified as Enterococcus faecalis strain NBRC 100481, show promise as in vitro probiotics for silkworm rearing, calling for further in vivo evaluation. Full article

To determine the evolution of microbial community and microbial shift under anaerobic processes, this study investigates the use of denaturing gradient gel electrophoresis (DGGE). In the DGGE, short- and medium-sized DNA fragments are separated based on their melting characteristics, and this technique is used in this study to understand the dominant bacterial community in mesophilic and thermophilic anaerobic digestion processes. Dairy manure is known for emitting greenhouse gases (GHGs) such as methane, and GHG emissions from manure is a biological process that is largely dependent on the manure conditions, microbial community presence in manure, and their functions. Additional efforts are needed to understand the GHG emissions from manure and develop control strategies to minimize the biological GHG emissions from manure. To study the microbial shift during anaerobic processes responsible for GHG emission, we conducted a series of manure anaerobic digestion experiments, and these experiments were conducted in lab-scale reactors operated under various temperature conditions (28 °C, 36 °C, 44 °C, and 52 °C). We examined the third variable region (V3) of the 16S rRNA gene fingerprints of bacterial presence in anaerobic environment by PCR amplification and DGGE separation. Results showed that bacterial community was affected by the temperature conditions and anaerobic incubation time of manure. The microbial community structure of the original manure changed over time during anaerobic processes, and the community composition changed substantially with the temperature of the anaerobic process. At Day 0, the sequence similarity confirmed that most of the bacteria were similar (>95%) to Acinetobacter sp. (strain: ATCC 31012), a Gram-negative bacteria, regardless of temperature conditions. At day 7, the sequence similarity of DNA fragments of reactors (28 °C) was similar to Acinetobacter sp.; however, the DNA fragments of effluent of reactors at 44 °C and 52 °C were similar to Coprothermobacter proteolyticus (strain: DSM 5265) (similarity: 97%) and Tepidimicrobium ferriphilum (strain: DSM 16624) (similarity: 100%), respectively. At day 60, the analysis showed that DNA fragments of effluent of 28 °C reactor were similar to Galbibacter mesophilus (strain: NBRC 10162) (similarity: 87%), and DNA fragments of effluent of 36 °C reactors were similar to Syntrophomonas curvata (strain: GB8-1) (similarity: 91%). In reactors with a relatively higher temperature, the DNA fragments of effluent of 44 °C reactor were similar to Dielma fastidiosa (strain: JC13) (similarity: 86%), and the DNA fragments of effluent of 52 °C reactor were similar to Coprothermobacter proteolyticus (strain: DSM 5265) (similarity: 99%). To authors’ knowledge, this is one of the few studies where DGGE-based approach is utilized to study and compare microbial shifts under mesophilic and thermophilic anaerobic digestions of manure simultaneously. While there were challenges in identifying the bands during gradient gel electrophoresis, the joint use of DGGE and sequencing tool can be potentially useful for illustrating and comparing the change in microbial community structure under complex anaerobic processes and functionality of microbes for understanding the consequential GHG emissions from manure. Full article