Isolation and optimization of the growth conditions of thermophilic microorganism from hot springs

Binh Chau hot spring locates in Bung Rieng commune, Xuyen Moc district, Ba Ria - Vung Tau province, and is the second hottest hot spring in Vietnam. Up to date, the microbial diversity of this hot spring has been mainly evaluated using the culture-dependent methods, thus, it could not be able to assess all of microorganisms there. In this report, the microbial community of Binh Chau hot spring was investigated by meta-analysis using Hiseq Illumina for sequencing. The results revealed that only 106,903 genes were annotated and 49,190 genes were unknown among 156,093 potential ORFs. The annotated genes consisted of 29,069 bacterial genes, 1,416 archaeal genes, 4 eukaryotic genes, 3 viral genes, and 76,411 unclassified genes. Among the annotated and classified genes, the microbial community of Binh Chau hot spring was characterized by the predominance of bacteria over archaea. At the phylum level, the most abundant phyla in Binh Chau hot spring were followed as Proteobacteria > Firmicutes > Cyanobacteria > Bacteroidetes > Planctomycetes > Thaumarchaeota > Actinobacteria > Euryarchaeota. These belonged mainly to the thermophilic group. A number of the phyla are reported to grow at high temperature, alkaline pH, and high salt concentrations. In addition to the culturable species, the metagenomic data is also indicated the presence of unculturable groups, for example phylum Acidobacteria and Chloroflexi of bacteria. The order Thermoplasmata, which is classified to class Thermoplasmatales of the phylum Euryarchaeota, as well as ultra-small cell and symbiotic archaea including phyla Parvarchaeota, Nanohaloarchaeota, and Nanoarchaeota were detected. The results in this study indicated an important insight into the microbial communities of Binh Chau hot spring, which revealed a promising thermophilic gene sources for their exploitation and conservation in Vietnam.

Hot Springs have unique geochemical features. Microorganisms-mediated arsenite oxidation is one of the major biogeochemical processes occurred in some hot springs. This study aimed to understand the diversities of genes and microorganisms involved in arsenite oxidation from the outlet of an untraversed hot spring located at an altitude of 4226 m. Microcosm assay indicated that the microbial community from the hot springwas able to efficiently oxidize As(III) using glucose, lactic acid, yeast extract or sodium bicarbonate as the sole carbon source. The microbial community contained 7 phyla of microorganisms, of which Proteobacteria and Firmicutes are largely dominant; this composition is unique and differs significantly from those of other described hot springs. Twenty one novel arsenite oxidase genes were identified from the samples, which are affiliated with the arsenite oxidase families of α-Proteobacteria, β-Proteobacteria or Archaea; this highlights the high diversity of the arsenite-oxidizing microorganisms from the hot spring. A cultivable arsenite-oxidizer Chelatococcu sp. GHS311 was also isolated from the sample using enrichment technique. It can completely convert 75.0 mg/L As(III) into As(V) in 18 days at 45 °C. The arsenite oxidase of GHS311 shares the maximal sequence identity (84.7%) to that of Hydrogenophaga sp. CL3, a non-thermotolerant bacterium. At the temperature lower than 30 °C or higher than 65 °C, the growth of this strain was completely inhibited. These data help us to better understand the diversity and functional features of the thermophilic arsenite-oxidizing microorganisms from hot springs.

Comparative evaluation of the biohydrogen production potential of thermophilic microorganisms isolated from hot springs located in Izmir

Thermophilic bacteria are biotechnologically important group of microorganisms due to their capability to produce a variety of thermostable enzymes, such as proteases. The aim of the present study was the optimization of protease production by thermophilic bacteria isolated from hot springs (54°C-55.5°C) in Maha Oya, Sri Lanka. Four isolates of thermophilic bacteria that belong to the genera Geobacillus, which were previously been isolated and identified, were used in the present investigation to study their protease production. Among the four isolates, Geobacillus toebii (DMBUK 107191) reached a maximum protein concentration with a level of 858 μg/mL and Geobacillus kaustophilus (DMBUK 107161) showed maximum protease activity of 2.232 units/mL (55°C, pH 7). Studies on the result of temperature and pH on protease activity discovered that the extreme protease activity was detected at 60°C over a pH range of 6-8. The isolates utilized several carbon and nitrogen sources for the production of proteases. Among the various carbon sources used, bacteria showed maximum protease activity in the presence of sucrose and fructose. Furthermore, bacteria showed a maximum yield of protease activity when gelatin was used as the nitrogen source.

H–O–C–S isotope and geochemical assessment of the geothermal area of Central Greece

The purpose of this study is screening and isolation of thermophilic microorganisms from a geothermal hot spring in Zharkent, Almaty region, Kazakhstan. The phylogenetic diversity and screening of theprokaryotic community thriving in Zharkent geothermal hot spring has been studied by microbiologicalmethods. The study confirmed that isolates from Zharkent geothermal hot spring are true thermophilesaccording to experiments and can be a source of thermostable enzymes; three isolates (AC1W, AC1S,AC4S) out of eight showed good enzymatic characteristics based on the results of their cultivation onselective media. Isolated thermophilic microorganisms, were cultivated under different conditions, suchas: temperature (50, 60, 70, 80, and 95оC), selective media (for the amylase, cellulase, and lipase producer), different pH (5, 6, 7, 8, and 10). Morphological features of the isolated bacterial strains wereexamined by scanning electron microscopy (SEM).Based on the data of biochemical tests and morphological characteristics (endospore formation,aerobic, positive for catalase and oxidase, optimal growth temperature of 75-85 °C and the optimal pHrange of growth is 7.5-8), isolated bacteria were presumably listed in the genus Geobacillus. Thus, ourresearch has shown that there is a rich variety of thermophilic microorganisms in Zharkent hot springs,which must be studied to obtain commercially important enzymes.Key words: hyperthermophiles, hydrolase, extremozymes, isolates

Thermophiles are microorganisms that inhabit at extremely high temperature environment. Thermoenzymes from thermophilic microorganisms are heat-stable, able to withstand denaturants of extremely acidic and alkaline conditions, and thus, have a considerable potential for many industrial applications. The aim of this study was to isolate and characterize amylolytic thermophiles. Two isolates of amylase producing thermophiles namely MA7K1T55C4 and MA8K2T55C8 were isolated previously from Dusun Tua Hot Spring, Selangor. The pH of the hot springs within the area was recorded to be slightly alkaline with temperature ranging from 55 ˚C to 70 ˚C. Two media were used to inoculate these samples, which were general enrichment medium, and selective medium that contains starch as the carbon source. Colonies with white abundant growth, irregular margin and glossy smooth were obtained from both samples. These isolates were subjected to gene amplification by PCR using a pair of universal primer to obtain their 16S rDNA gene. Obtained gene was sequenced and analyzed using bioinformatics tools. They were found to be from the genus Brevibacillus, spore-forming type; Gram's positive, aerobic, rod-shaped and capable of producing amylase. Bioinformatics analysis shown that isolate MA7K1T55C4 and isolate MA8K2T55C8 had the highest homology with the Brevibacillus berstelensis with the score of 99%.

Environments with temperatures from 50°C to 80°C are rare in nature and are almost exclusively associated with geothermal regions including hot springs, solar-heated soils and volcanic areas. Thermophilic bacteria already exist and prefer in such habitats. Since innate tolerance to thermal environment and potential chassis for extracellular enzymes such as lipase, protease and amylase, which are utilized widely in the industrial fermentation, thermophilic bacteria have been becoming one of the objects for microbiologists worldwide, recently. This study aimed to isolate and identify thermophilic bacteria from hot springs in several provinces in Vietnam such as Ba Ria - Vung Tau and Khanh Hoa. In the results, six moderate thermophilic bacterial strains (namely BM7, BS5, NS1, NS3, NS4, and NW6) that could grow at 55oC were purified from the hot spring ecosystems. All micro morphology of isolates were recorded as rod-shaped, Gram positive, and endospore forming. The results of 16S rDNA sequencing and phylogenetic analysis showed that these isolate belonged to group I of Bacillus genus (the thermophilic group). The isolated strains NS1, NS3, NS4, BS5, NW6 and BM7 were identified to belong to the Bacillus genus, species as Bacillus sp. Resulting strains are potential candidates for industrial applications due to its stable fitness in a hash environment, particularly at high temperature. In addition, this study provides a useful insight into the diverse community of thermophilic bacteria (Bacillus spp.) in several hot springs of Vietnam, that can be applied as bacterial cell factories to produce biomaterials, biofuels, or valuable compounds in the future.

The goal of the work was to study the effect of addition of organic carbon source required for microorganisms oxidizing sulfide minerals in the nutrient medium on the rate of copper concentrate bioleaching. The concentrate containing 31.6% Fe, 19.2% Cu, and 2.9% Zn was bioleached in a batch mode for 30 days using mixed culture of acidophilic microorganisms. In the first reactor, biooxidation was performed at 45°C without additional carbon sources. In the second and in the third reactors, biooxidation was performed at 45°C and 55°C and nutrient medium was supplemented with 0.02% of yeast extract. Biooxidation without yeast extract allowed extracting 47 and 92% of copper and zinc. During biooxidation at 45°C using the medium supplemented with yeast extract, 54 and 94% of copper and zinc were extracted. During bioleaching at 55°C, 48% and 84% of copper and zinc were extracted. Thus, yeast extract allowed increasing copper bioleaching rate at 45°C, but temperature increase up to 55°C led to decrease in bioleaching efficiency. It may be explained by the fact that yeast extract increased activity of acidophilic microorganisms, which may consume organic carbon sources, while high temperature may inhibit some microorganisms and, therefore, does not allow increasing bioleaching rate.

Thermophiles are microorganisms that inhabit at extremely high temperature environment. Thermoenzymes from thermophilic microorganisms are heat-stable, able to withstand denaturants of extremely acidic and alkaline conditions, and thus, have a considerable potential for many industrial applications. Thermophilic microorganisms are able to degrade on certain substrates and its subsequent utilization are said to be important for global carbon sources. The aim of this study was to screen and isolate the thermophiles capable of producing thermostable lipase, amylase and cellulase. The highly sedimented water sample was taken from the local hot spring located at Hulu Langat, Selangor. The pH of the hot springs within the area was recorded to be slightly alkaline with the temperature ranging from 55°C to 70°C. The results of this study showed five bacterial strains with positive results for lipolytic activity via screening on selective medium containing Tween 80, 11 positive isolates of amylase-producing thermophiles that displayed large and distinct halo-forming zone upon the treatment of flooding iodine method on starch-nutrient medium and 5 positive isolates of cellulolytic thermophiles screened via selective carboxymethylcellulose (CMC) medium. These positive isolates were further characterized and identified using morphological characterization techniques and conventional biochemical tests.

Springs on the eastern and western shores of Lake Natron Basin (LNB), located within the eastern branch of the East Africa Rift System (EARS) in Northern Tanzania had a discharge temperature that ranged between 34.0 °C and 51.2 °C, while the pH varied from 8.0 to 10.7. The electrical conductivity (EC) ranged between 5,007 µS/cm and 49,200 µS/cm. Cold waters had a temperature of 31.9 °C to 32.5 °C, while the pH ranged between 8.0 and 8.3, and the EC ranged between 1,401 µS/cm and 3,806 µS/cm. The stable isotope composition varied between -2.4 ‰ and -5.3 ‰ for δ18O, and -15.5 ‰ to -29.3 ‰ for δ2H. The isotopic composition of thermal and cold water of LNB indicates a significant contribution of meteoric water in the recharge of the hydrothermal system. However, thermal water is affected by evaporation, water-rock interaction, carbon dioxide (CO2) exchange and condensation processes. Tritium analysis indicated that the spring water in the LNB hydrothermal system has a residence time of more than 50 years. Keywords: thermal water; Lake Natron Basin; stable isotopes; springs.

In the present study, the growth and total lipid contents of two oleaginous fungal isolates Aspergillus terreus, Aspergillus fumigatus were compared in different nitrogen and organic carbon sources. Artificially the fungi were cultured on media consisting of various mono- or di- or polysaccharides and peptone or yeast extract as elementary sources for carbon and nitrogen, respectively. Media containing sucrose /yeast extract or glucose/ yeast extract were the most effective for lipid production from fungal, during two weeks incubation period, the highest biomass of dry weight was (19.6 , 18.8) g / L , (25.8 , 30.5) g /L and lipid yield (1, 0.97 )g/L, (0.65, 0.65) g/ L for two isolates Aspergillus terreus and Aspergillus fumigatus respectively then followed maltose/ yeast extract then starch / yeast extract .Analysis of fatty acid produced by two fungal isolates using Gas Chromatography showed the existence of a wide range of fatty acids, these fatty acids were together saturated and unsaturated. The results revealed that the unevenness in the fatty acids composition mainly depends on the type of carbon and nitrogen sources. The existence of saturated and mono saturated fatty acids in A.fumigatus, A. terreus prove that they are good candidate for biodiesel production. The analysis of fatty acid from the fungal growth showed the existence of a wide range of fatty acids Palmitic ,Oleic acids , stearic acid and linoleic acid together saturated and unsaturated. The results revealed that the unevenness in the fatty acid composition mainly depends on the type of carbon and nitrogen sources. The presence of saturated and mono saturated fatty acid in A.fumigatus , A. terreus prove that it is a good candidate for biodiesel production.

Source and sequestration of sediment organic carbon from different elevation zones in estuarine wetland, North China

1) When calcium carbonate was formed in the laboratory from the calcium bicarbonate solution through which carbon dioxide gas had not been bubbled during the process of the calcium carbonate formation: a) Only calcite was formed at a temperature below 25°C; aragonite was formed above 25°C, and vaterite was formed above 40°C. Between 40 and 60°C a large amount of vaterite was formed. With a rise in the temperature of the mother solution, the proportion of calcite decreased, b) Vaterite was easily formed from the calcium bicarbonate solution containing sodium chloride at a high temperature. With an increase in the temperature and in sodium chloride concentration, the proportion of vaterite increased, c) With an increase in both temperature and magnesium chloride concentration, the proportion of aragonite increased but that of calcite decreased. The presence of magnesium chloride in the mother solution hindered the formation of vaterite. 2) When calcium carbonate was formed in the laboratory from the calcium bicarbonate solution containing sodium chloride (or magnesium chloride) and bubbled with carbon dioxide gas continuously during the calcium carbonate formation, only calcite was formed despite a high temperature. 3) When calcium carbonate was deposited in thermal spring waters of a high temperature, aragonite was formed. However, from the waters into which carbon dioxide gas was supplied, in other words, from the acidic solution, calcite was formed despite a high temperature, and aragonite was formed from the waters to which carbon dioxide was not supplied, in other words, from the basic waters, despite a low temperature. 4) Up to the present time, vaterite has not been found in the deposits of thermal springs because: a) Calcium carbonate deposits of thermal springs were either formed at a location where carbon dioxide gas was supplied to the mother waters or where the concentration of magnesium ions in the waters was rather high, or b) even if vaterite had been formed, it would have changed to calcite or aragonite very quickly because of its low stability in solution. 5) In spring waters with the same chemical constituents, pH value, and temperature, the proportion of aragonite increased and that of calcite decreased with an increase in the rate of carbon dioxide gas escape from the waters and in the rate of calcium carbonate formation. 6) Aragonite was readily found in calcium carbonate precipitates formed floating on the surface of thermal spring waters, but it was not likely to be found in the precipitates deposited on the wall or bottom in the same waters. The polymorphic formation of calcium carbonate formed in thermal springs depends mainly on the temperature, the concentrations of sodium ions, magnesium ions and carbon dioxide (pH) in the waters, and on the rate of calcium carbonate formation when calcium carbonate is formed from a similar water.

Andalas (Morus macroura Miq.) is one of the endemic plants in West Sumatera, which is known to have bioactive compounds that have the potential as antifungal. One of the effective ways to produce bioactive compounds from the plants is by utilizing endophytic bacteria through a fermentation process. One of the factor that influence fermentation is the medium. The purpose of this study was to see the effect of carbon and organic nitrogen sources on the growth of the Andalas endophytic bacteria ATB 10-6 isolate and its ability to produce antifungal compounds. The carbon sources used were glucose, lactose, maltose, sucrose, and starch. The types of organic nitrogen sources used were yeast extract, malt extract, beef extract and peptone water. Bacterial growth was observed by measuring optical density (OD), while antifungal activity was carried out by diffusion method. The result showed that a starch and yeast extract was the best carbon and organic nitrogen for the Andalas endophytic bacteria growth. These two types of nutrients are also the best sources of organic carbon and nitrogen to produce antifungal compounds.