Molecular identification of amylase-producing thermophilic bacteria isolated from Bukit Gadang Hot Spring, West Sumatra, Indonesia

Abstract. Ardhi A, Sidauruk AN, Suraya N, Pratiwi NW, Pato U, Saryono. 2020. Molecular identification of amylase-producing thermophilic bacteria isolated from Bukit Gadang Hot Spring, West Sumatra, Indonesia. Biodiversitas 21: 994-1000. Amylase is one of the hydrolytic enzymes that is widely used in a wide number of industrial processes such as food, fermentation, textile, paper, detergent, and pharmaceutical industries. Amylase produced by thermophilic bacteria may be thermostable, which is very beneficial in several applications requiring high temperature, for example, the process of gelatinization, liquefaction, and saccharification are performed in high temperature involved in the starch processing. In this study, the amylase-producing ability of thermophilic bacteria isolated from Bukit Gadang hot spring, West Sumatra, Indonesia, was checked and followed by molecular identification. Thirteen isolates that were successfully isolated from the hot springs were microscopically and macroscopically characterized, biochemically tested, and determined their amylase enzyme activity both qualitatively and quantitatively. The isolate that performed the best amylase activity was identified using the molecular technique. The DNA sequencing was carried out in 16S rRNA and continued with BLAST search for species identification. The result of molecular identification showed that the isolate with the best amylase activity was identified as Bacillus licheniformis. The optimum amylase production (231.33 U/ml) and the best enzyme-specific activity (101.79 U/mg) were obtained at the incubation time of 36 hours.

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Isolation and molecular identification of carbohydrase and protease producing Bacillus subtilis JCM 1465 isolated from Penen Hot Springs in North Sumatra, Indonesia

Biodiversitas Journal of Biological Diversity ◽

10.13057/biodiv/d201205 ◽

2019 ◽

Vol 20 (12) ◽

Author(s):

Edy Fachrial ◽

Sari Anggraini ◽

Harmileni ◽

Titania T Nugroho ◽

Saryono

Keyword(s):

Bacillus Subtilis ◽

Molecular Identification ◽

Hot Springs ◽

Hot Spring ◽

Thermophilic Bacteria ◽

Temperature Stability ◽

Subtilis Strain ◽

Clear Zone ◽

Promising Source ◽

North Sumatra

Abstract. Fachrial E, Anggraini S, Harmileni, Nugroho TT, Saryono. 2019. Isolation and molecular identification of carbohydrase and protease producing Bacillus subtilis JCM 1465 isolated from Penen Hot Springs in North Sumatra, Indonesia. Biodiversitas 20: 3493-3498. The application of enzymes industrially is increasing every year and thermophilic microbes are a promising source of these enzymes for industrial use due to their temperature stability. The aim of this study, therefore, was to isolate, characterize and identify the enzyme producer-thermophilic bacteria from the Penen Hot Spring in Deli Serdang, North Sumatra, Indonesia. In the experimental setup, carbohydrase activities including inulinase and amylase were determined by the formation of clear zone around the colonies after soaking with Lugol on the TSA medium supplemented with 1% inulin powder and 1% starch. Similarly, the protease activities were determined by the formation of clear zone around the colonies on Skim Milk Agar Medium. Then, of the 11 isolates, only one known as UTMP 12 showed maximum enzyme activity. The isolate was then characterized based on morphology and biochemistry and found to be Bacillus subtilis strain JCM 1465 (accession number NR_113265, homolog 99.72%). Furthermore, this is the first study on carbohydrase and protease activities of Bacillus subtilis strain JCM 1465 and the result shows that the thermophilic bacteria are needed in the production of carbohydrase and protease.

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Molecular identification of cellulase and protease producing Bacillus tequilensis UTMSA14 isolated from the geothermal hot spring in Lau Sidebuk Debuk, North Sumatra, Indonesia

Biodiversitas Journal of Biological Diversity ◽

10.13057/biodiv/d211035 ◽

2020 ◽

Vol 21 (10) ◽

Author(s):

Edy Fachrial ◽

Raden Roro Jenny Satyo Putri ◽

I Nyoman Ehrich Lister ◽

Sari Anggraini ◽

Harmileni HARMILENI ◽

...

Keyword(s):

Phylogenetic Analysis ◽

Molecular Identification ◽

Hot Springs ◽

Hot Spring ◽

Thermophilic Bacteria ◽

Skim Milk ◽

Industrial Applications ◽

Accession Number ◽

Bacillus Tequilensis ◽

North Sumatra

Abstract. Fachrial E, Putri RRJS, Lister INE, Anggraini S, Harmileni, Nugroho TT, Saryono. 2020. Molecular identification of cellulase and protease producing Bacillus tequilensis UTMSA14 isolated from the geothermal hot spring in Lau Sidebuk Debuk, North Sumatra, Indonesia. Biodiversitas 21: 4719-4725. The industrial need for stable microbial enzymes tends to increase every year. The aim of this study was to isolate and identify the protease and cellulase producing thermophilic bacteria isolated from the geothermal spring of Lau Sidebuk Debuk, North Sumatra, Indonesia. The protease activity was determined based on the formation of halo zone on Skim Milk Agar, while cellulase was confirmed using CMC plate agar method. Nine bacterial isolates were successfully obtained from the sediments and water, and both activities were observed in only one isolate, known as UTMSA14. These isolates were characterized biochemically and morphologically. The molecular identification of UTMSA14 was done by 16S rRNA sequencing based on BLAST and phylogenetic analysis using MEGA X. Phylogenetic analysis showed that UTMSA14 clustered together with Bacillus tequilensis strain 10b (accession number NR_104919.1), which ascertained the isolate as Bacillus tequilensis strain 10b (accession number NR_104919.1). This is the first report of a thermophilic bacteria isolated from Lau Sidebuk Debuk hot springs, with the ability to produce cellulase and protease. further research is needed to purify and characterize the resulting enzymes, with a potential for industrial applications.

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Identification, Characterization, and Hydrolase Producing Performance of Thermophilic Bacteria: Geothermal Hot Springs in Eastern and Southeastern Anatolia Region of Turkey

10.21203/rs.3.rs-348608/v1 ◽

2021 ◽

Author(s):

Orhan Uluçay ◽

Arzu Görmez ◽

Cem Öziç

Keyword(s):

Hot Springs ◽

Hot Spring ◽

Thermophilic Bacteria ◽

Hydrolytic Enzymes ◽

Bacillus Coagulans ◽

Rrna Gene ◽

Bacterial Isolates ◽

Thermophilic Bacilli ◽

Geobacillus Kaustophilus ◽

The Rich

Abstract In the last twenty years, researchers have increasingly focused on the rich microorganism-based diversity of natural hot spring resources to explore the benefits of thermophiles in industrial and biotechnological fields. For this purpose, a total of 83 thermophilic bacilli were isolated from 7 different geothermal hot springs (at temperatures ranging between 40 and 85 ◦ C) located in the east and Southeastern of Turkey. These isolates were identified by different methods such as physiological, morphological, biochemical, molecular properties. According to 16S rRNA gene sequence analysis, 5 different isolates ( Bacillus coagulans , Bacillus licheniformis , Bacillus subtilis , Bacillus thuringiensis , and Geobacillus kaustophilus ) were identified. It was observed that B. licheniformis and B. subtilis were the most species obtained from the researched hot spring sources. Phylogenetic relationships of isolates were evaluated with the help of a phylogenetic tree. The conditions of bacterial isolates to synthesize various hydrolytic enzymes such as protease, cellulase, lipase, and amylase were investigated. When the potential of isolates to produce hydrolytic enzymes was examined, protease 73 (88%), cellulase 34 (41%), lipase 69 (83%), and amylase 68 (82%) were detected. All isolates have at least one or more extracellular protease, cellulase, amylase, or lipase activity. Besides, 32.8% (27) of bacterial isolates were able to synthesize all of the hydrolytic enzymes.

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Carbon Source Preference in Chemosynthetic Hot Spring Communities

Applied and Environmental Microbiology ◽

10.1128/aem.00511-15 ◽

2015 ◽

Vol 81 (11) ◽

pp. 3834-3847 ◽

Cited By ~ 20

Author(s):

Matthew R. Urschel ◽

Michael D. Kubo ◽

Tori M. Hoehler ◽

John W. Peters ◽

Eric S. Boyd

Keyword(s):

High Temperature ◽

National Park ◽

Hot Springs ◽

Dissolved Inorganic Carbon ◽

Hot Spring ◽

Affinity Constant ◽

Rrna Gene ◽

Organic Substrates ◽

Short Term ◽

Acetate Assimilation

ABSTRACTRates of dissolved inorganic carbon (DIC), formate, and acetate mineralization and/or assimilation were determined in 13 high-temperature (>73°C) hot springs in Yellowstone National Park (YNP), Wyoming, in order to evaluate the relative importance of these substrates in supporting microbial metabolism. While 9 of the hot spring communities exhibited rates of DIC assimilation that were greater than those of formate and acetate assimilation, 2 exhibited rates of formate and/or acetate assimilation that exceeded those of DIC assimilation. Overall rates of DIC, formate, and acetate mineralization and assimilation were positively correlated with spring pH but showed little correlation with temperature. Communities sampled from hot springs with similar geochemistries generally exhibited similar rates of substrate transformation, as well as similar community compositions, as revealed by 16S rRNA gene-tagged sequencing. Amendment of microcosms with small (micromolar) amounts of formate suppressed DIC assimilation in short-term (<45-min) incubations, despite the presence of native DIC concentrations that exceeded those of added formate by 2 to 3 orders of magnitude. The concentration of added formate required to suppress DIC assimilation was similar to the affinity constant (Km) for formate transformation, as determined by community kinetic assays. These results suggest that dominant chemoautotrophs in high-temperature communities are facultatively autotrophic or mixotrophic, are adapted to fluctuating nutrient availabilities, and are capable of taking advantage of energy-rich organic substrates when they become available.

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Short Communication: The emergence and rise of indigenous thermophilic bacteria exploration from hot springs in Indonesia

Biodiversitas Journal of Biological Diversity ◽

10.13057/biodiv/d211156 ◽

2020 ◽

Vol 21 (11) ◽

Author(s):

Kenny Lischer ◽

ANANDA BAGUS RICHKY DIGDAYA PUTRA ◽

Brian Wirawan Guslianto ◽

Forbes Avila ◽

Sarah Grace Sitorus ◽

...

Keyword(s):

High Temperature ◽

16S Rrna ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Short Communication ◽

Hot Springs ◽

Thermophilic Bacteria ◽

Whole Genome ◽

Thermostable Enzymes ◽

The Pacific

Abstract. Lischer K, Putra ABRD, Guslianto BW, Avilla F, Sitorus SG, Nugraha Y, Sarmoko. 2020. Short Communication: The emergence and rise of indigenous thermophilic bacteria exploration from hot springs in Indonesia. Biodiversitas 21: 5474-5481. Indonesia is an archipelagic country located in the pacific ring of fire, and is estimated to cause numerous hot springs spread across the country. In addition, small living microbes have been explored in these locations since 1985. These microbes possess the ability to survive in areas with high temperature (more than 40oC-90oC), and are therefore termed thermophiles. Hence, massive explorations have been conducted on Java island and other unexplored areas at Sumatra to Papua in New Guinea islands. Moreover, a total of 71 hot springs characterized by the presence of thermophilic bacteria have been explored in Indonesia. These investigations ensue with various approaches, including through conventional and microbiological, 16S rRNA, as well as whole-genome sequencing methods. In addition to species exploration, the application of thermophiles has become a topic of interest from 1999, especially based on thermostable enzymes with the capacity to maintain activity at high-temperature conditions. These include amylase, protease, lipase, xylanase, esterase, and cellulase as the most common isolated form, which indicates the existence of significant extractable potentials. Hence, there is a need for further research in terms of both exploration and application purposes.

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Anoxybacillus sp. AH1, an α-amylase-producing thermophilic bacterium isolated from Dargeçit hot spring

Biologia ◽

10.1515/biolog-2015-0111 ◽

2015 ◽

Vol 70 (7) ◽

Cited By ~ 3

Author(s):

Ömer Acer ◽

Hemşe Pırınççiğlu ◽

Fatma Matpan Bekler ◽

Reyhan Gül-Güven

Keyword(s):

Hot Springs ◽

Hot Spring ◽

Thermophilic Bacteria ◽

Potato Starch ◽

Nitrogen Sources ◽

Soluble Starch ◽

Thermophilic Bacterium ◽

Carbon And Nitrogen Sources ◽

Carbon And Nitrogen ◽

Optimum Growth Temperature

AbstractThe present study was conducted to isolate α-amylase-producing thermophilic bacteria from Darge¸cit hot springs in Turkey. The morphological, biochemical and physiological characterisation, as well as genetic analysis by 16S rRNA sequences indicated that the isolated strain AH1 was a member of Anoxybacillus genus. The strain was aerobe, Gram-positive and spore-forming rod, exhibiting optimum growth temperature and pH of 60ºC and 7.0-7.5, respectively. Optimization of growth medium and enzyme assay conditions for extracellular α-amylase production by the novel thermophilic Anoxybacillus sp. AH1 were carried out in many different media containing a variety of carbon and nitrogen sources. Among various carbon and nitrogen sources, peptone (2054.1 U/mL) at 1% and maltose (1862.9 U/mL) at 0.5% increased α-amylase activity, compared to controls. Moreover, a high enzyme production was observed with potato starch at 0.5% and 1% (2668.4 U/mL and 3627 U/mL, respectively), as well as with 1% soluble starch (2051.9 U/mL). The enzyme activity was found to be rather high in the presence of CaCl

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Diversity analysis and metagenomic insights into the Antibiotic Resistance and Metal Resistances among Hot Spring Bacteriobiome-insinuating inherent environmental baseline levels of Antibiotic and Metal tolerance

10.1101/773788 ◽

2019 ◽

Author(s):

Ishfaq Nabi Najar ◽

Mingma Thundu Sherpa ◽

Sayak Das ◽

Nagendra Thakur

Keyword(s):

Antibiotic Resistance ◽

Resistance Genes ◽

Hot Springs ◽

Metal Tolerance ◽

Hot Spring ◽

Thermophilic Bacteria ◽

Antibiotic Resistance Genes ◽

Metal Resistance ◽

Culture Independent ◽

Maximum Similarity

AbstractMechanisms of occurrence and expressions of antibiotic resistance genes (ARGs) in thermophilic bacteria are still unknown owing to limited research and data. The evolution and proliferation of ARGs in the thermophilic bacteria is unclear and needs a comprehensive study. In this research, comparative profiling of antibiotic resistance genes and metal tolerance genes among the thermophilic bacteria has been done by culture-independent functional metagenomic methods. Metagenomic analysis showed the dominance of Proteobacteria, Actinobacteria. Firmicutes and Bacteroidetes in these hot springs. ARG analysis through shotgun gene sequencing was found to be negative in case of thermophilic bacteria. However, few of genes were detected but they were showing maximum similarity with mesophilic bacteria. Concurrently, metal resistance genes were also detected in the metagenome sequence of hot springs. Detection of metal resistance gene and absence of ARG’s investigated by whole genome sequencing, in the reference genome sequence of thermophilic Geobacillus also conveyed the same message. This evolutionary selection of metal resistance over antibiotic genes may have been necessary to survive in the geological craters which are full of different metals from earth sediments rather than antibiotics. Furthermore, the selection could be environment driven depending on the susceptibility of ARG’s in thermophilic environment as it reduces the chances of horizontal gene transfer. With these findings this article highlights many theories and culminates different scopes to study these aspects in thermophiles.

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Distinct microbial composition and functions in an underground high-temperature hot spring at different depths

10.5194/bg-2019-406 ◽

2019 ◽

Cited By ~ 1

Author(s):

Shijie Bai ◽

Xiaotong Peng

Keyword(s):

High Temperature ◽

Microbial Communities ◽

Hot Springs ◽

Hot Spring ◽

Hydrogen Oxidation ◽

Archaeal Community ◽

Limited Area ◽

Rrna Gene ◽

Microbial Composition ◽

Different Depths

Abstract. The microbial diversity and functions of three high-temperature neutral hot springs water samples at different depths (0 m, 19 m and 58 m) were investigated based on 16S rRNA gene sequencing and a functional gene array (GeoChip 5.0). The results revealed that the bacterial communities were distinct at different depths in the hot springs. Additionally, in response to the depths, bacterial/archaeal community compositions exhibited shifts over the depth profiles. Aquificae, Alpha-proteobacteria, and Deinococcus-Thermus were the dominating phyla at 0 m, 19 m, and 58 m, respectively. Hydrogenobacter, Sphingobium, and Thermus were the most abundant genera at 0 m, 19 m, and 58 m, respectively. The phylum Thaumarchaeota was the most abundant member of the archaeal community in the samples at different hot spring depths. Functional results of the microbial communities indicated that microbial metabolic functions were mainly related to sulfur, nitrogen cycling, and hydrogen oxidation. In summary, our results demonstrated that distinct microbial communities and functions were found at different depths of hot springs in a very limited area. These findings will provide new insights into the deep-subsurface biosphere associated with terrestrial hot springs.

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Characterizing and Evaluating the Diverse Microbial Communities and Their Mercury Resistance Potential from Hot Spring Sites Representing Gradients in Temperature and pH in Yellowstone National Park

Aresty Rutgers Undergraduate Research Journal ◽

10.14713/arestyrurj.v1i1.137 ◽

2020 ◽

Vol 1 (1) ◽

Author(s):

Yelizaveta Rassadkina ◽

Spencer Roth ◽

Tamar Barkay

Keyword(s):

Microbial Communities ◽

Yellowstone National Park ◽

National Park ◽

Hot Springs ◽

Hot Spring ◽

Thermophilic Bacteria ◽

Sequence Data ◽

Mercury Resistance ◽

Sample Site ◽

Microorganism Community

Yellowstone National Park is home to many different hot springs, lakes, geysers, pools, and basins that range in pH, chemical composition, and temperature. These different environmental variations provide a broad range of conditions that select and grow diverse communities of microorganisms. In this study, we collected samples from geochemically diverse lakes and springs to characterize the microbial communities present through 16S rRNA metagenomic analysis. This information was then used to observe how various microorganisms survive in high mercury environments. The results show the presence of microorganisms that have been studied in previous literature. The results also depict gradients of microorganisms including thermophilic bacteria and archaea that exist in these extreme environments. In addition, beta diversity analyses of the sequence data showed site clustering based primarily on temperature instead of pH or sample site, suggesting that while pH, temperature, and sample site were all shown to be significant, temperature is the strongest factor driving microorganism community development. While it is important to characterize the microorganism community present, it is also important to understand how this community functions as a result of its selection. Along with looking at community composition, genomic material was tested to see if it contained mercury methylating (hgcA) or mercury reducing (merA) genes. Out of 22 samples, three of them were observed to have merA genes, while no samples had hgcA genes. These results indicate that microorganisms in Mustard and Nymph Springs may use mercury reduction. Understanding how microorganisms survive in environments with high concentrations of toxic pollutants is crucial because it can be used as a model to better understand mechanisms of resistance and the biogeochemical cycle, as well as for bioremediation and other solutions to anthropogenic problems.

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ISOLATION OF THERMOPHILIC LIPASE PRODUCING BACTERIUM FROM HOT SPRINGS AT THE EAST COAST OF PENINSULAR MALAYSIA

Journal of Tropical Life Science ◽

10.11594/jtls.11.01.01 ◽

2021 ◽

Vol 11 (1) ◽

pp. 1-10

Author(s):

Tengku Hamid ◽

◽

Nur Abidin ◽

Nurmusfirah Hasan ◽

◽

...

Keyword(s):

Hot Springs ◽

East Coast ◽

Hot Spring ◽

Thermophilic Bacteria ◽

Natural Habitat ◽

Primary Source ◽

Industrial Applications ◽

Peninsular Malaysia ◽

Thermostable Enzymes ◽

Thermophilic Microorganisms

Hot spring is a natural habitat for thermophilic bacteria and the primary source of thermostable enzymes useful in industrial applications. In Malaysia, the search for thermophilic organisms has been focused on hot springs, especially on the peninsular West coast. In this work, lipase or esterase producing thermophilic microorganisms were isolated from East coast hot springs in Pahang and Terengganu's states. Morphological and biochemical analysis were carried out on Isolates LH1, LH2, LH3, LH4, LH5, B2B2 and S1B4, which showed that they are gram positive, aerobic, spore forming, and motile organisms. All of the seven isolates showed the ability to grow at 45°C and formed hydrolysis zones on tributyrin agar plates. However, only isolate B2B2 and S1B4 were able to thrive at higher temperatures of up to 65°C. The genotypic characterisation was carried out using 16S rRNA sequencing. Bacillus and Geobacillus species were found to be the dominant bacteria isolated from these hot springs. From La hot spring, isolate LH1 (MT 645486), Isolates LH2 (MT645483), LH3 (MT645484), LH4 (MT 645485) and LH5 (MT 645487) were all closely related to Bacillus sp. (at 97.3-97.9%). Meanwhile, from Bentong and Sungai Lembing hot springs, isolates B2B2 (MT668631) and S1B4 (MT668632) were near related to either Geobacillus kaustophilus or Geobacillus thermoleovorans; each at 98.5% and 97.9% similarity, respectively. These strains from Geobacillus sp. were able to thrive at higher temperature and their thermostable esterases or lipases have properties useful for biotechnological applications.

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