Thermophilic Research Articles

Enhanced biohydrogen production from high loads of unpretreated cattle manure by cellulolytic bacterium Caldicellulosiruptor bescii at 75 °C

Caldicellulosiruptor bescii is the most thermophilic cellulolytic bacterium capable of fermenting crystalline cellulose identified to date, and it also has a superior ability to degrade plant biomass without any pretreatment. This study is the first to assess the potential of utilizing unpretreated cattle manure (UCM) as a feedstock for hydrogen (H2) production by C. bescii at a concentration range between 2.5–50 g volatile solids (VS)/L. At 50 g VS/L UCM concentrations, H2 production ceased due to inhibition of C. bescii. To alleviate the impacts of inhibition, two strategies were adopted: (i) reduction of H2 build-up in the reactor headspace via gas sparging and (ii) adaptation of C. bescii to UCM via adaptive laboratory evolution (ALE). The former increased H2 yield by 47% compared to the control reactors, where no sparging was applied. The latter increased H2 yield by 142% compared to the control reactors inoculated by the wild type C. bescii. The UCM-adapted C. bescii demonstrated a remarkable H2 yield of 161.3 ± 1.6 mL H2/g VSadded at 15 g VS/L. This yield represents a twofold increase compared to the maximum H2 yield reported in the literature amongst fermentation studies utilizing manure as feed. At 15 g VS/L, around 73% of UCM was solubilized, and the carbon balance indicated that most of the effluent carbon was in the sugar- and acid-form. The remarkable ability of C. bescii to produce H2 from UCM under non-sterile conditions presents a significant potential for sustainable biohydrogen production from renewable feedstocks.

Isolation and Characterization of Thermus thermophilus Strain ET-1: An Extremely Thermophilic Bacterium with Extracellular Thermostable Proteolytic Activity Isolated from El Tatio Geothermal Field, Antofagasta, Chile.

The present study describes the isolation of an extremely thermophilic bacterium from El Tatio, a geyser field in the high planes of Northern Chile. The thermophile bacterium named Thermus thermophilus strain ET-1 showed 99% identity with T. thermophilus SGO.5JP 17-16 (GenBank accession No. CP002777) by 16S rDNA gene analysis. Morphologically, the cells were non-sporeforming Gram-negative rods that formed colonies with yellow pigmentation. This strain is able to proliferate between 55 and 80 °C with a pH range of 6-10, presenting an optimum growth rate at 80 °C and pH 8. The bacterium produces an extracellular protease activity. Characterization of this activity in a concentrated enzyme preparation revealed that extracellular protease had an optimal enzymatic activity at 80 °C at pH 10, a high thermostability with a half-life at 80 °C of 10 h, indicating that this enzyme can be classified as an alkaline protease. The proteolytic enzyme exhibits great stability towards chelators, divalent ions, organic solvents, and detergents. The enzyme was inhibited by phenylmethylsulfonyl fluoride (PMSF), implying that it was a serine protease. The high thermal and pH stability and the resistance to chelators/detergents suggest that the protease activity from this T. thermophilus. strain could be of interest in biotechnological applications.

Characterization of the Pro101Gln mutation that enhances the catalytic performance of T. indicus NADH-dependent d-lactate dehydrogenase

NADH-dependent d-lactate dehydrogenases (d-LDH) are important for the industrial production of d-lactic acid. Here, we identify and characterize an improved d-lactate dehydrogenase mutant (d-LDH1) that contains the Pro101Gln mutation. The specific enzyme activities of d-LDH1 toward pyruvate and NADH are 21.8- and 11.0-fold greater compared to the wild-type enzyme. We determined the crystal structure of Apo-d-LDH1 at 2.65Å resolution. Based on our structural analysis and docking studies, we explain the differences in activity with an altered binding conformation of NADH in d-LDH1. The role of the conserved residue Pro101 in d-LDH was further probed in site-directed mutagenesis experiments. We introduced d-LDH1 into Bacillus licheniformis yielding a d-lactic acid production of 145.9g L-1 within 60h at 50°C, which was three times higher than that of the wild-type enzyme. The discovery of d-LDH1 will pave the way for the efficient production of d-lactic acid by thermophilic bacteria.

A Method for Rapid Polyethyleneimine-Based Purification of Bacteriophage-Expressed Proteins from Diluted Crude Lysates, Exemplified by Thermostable TP-84 Depolymerase.

Purification of bacteriophage-expressed proteins poses methodological difficulties associated with the need to process entire culture medium volume upon bacteriophage-induced bacterial cell lysis. We have used novel capsule glycosylase-depolymerase (TP84_26 GD) from bacteriophage TP-84, infecting thermophilic Geobacillus stearothermophilus bacteria, as a representative enzyme to develop a method for rapid concentration and purification of the enzyme present in diluted crude host cell lysate. A novel variant of the polyethyleneimine (PEI)-based purification method was devised that offers a fast and effective approach for handling PEI-facilitated purification of bacteriophage-expressed native proteins. Due to the very basic nature of PEI, the method is suitable for proteins interacting with nucleic acids or acidic proteins, where either mixed PEI-DNA or RNA-protein complexes or PEI-acidic protein complexes are reversibly precipitated. (i) The method is of general use, applicable with minor modifications to a variety of bacteriophage cell lysates and proteins. (ii) In the example application, TP84_26 GD was highly purified (over 50%) in a single PEI step; subsequent chromatography yielded a homogeneous enzyme. (iii) The enzyme's properties were examined, revealing the presence of three distinct forms of the TP84_26 GD. These forms included soluble, unbound proteins found in host cell lysate, as well as an integrated form within the TP-84 virion.

Initial characterization of an iron superoxide dismutase from Thermobifida fusca

Superoxide dismutases (SODs) are enzymes that catalyze the dismutation of the superoxide radical anion into O2 and H2O2 in a two-step reaction. They are ubiquitous to all forms of life and four different types of metal centers are detected, dividing this class of enzymes into Cu-/Zn-, Ni-, Mn-, and Fe-SODs. In this study, a superoxide dismutase from the thermophilic bacteria Thermobifida fusca (TfSOD) was cloned and expressed before the recombinant enzyme was characterized. The enzyme was found to be active for superoxide dismutation measured by inhibition of cytochrome c oxidation and the inhibition of the autoxidation of pyrogallol. Its pH-optimum was determined to be 7.5, while it has a broad temperature optimum ranging from 20 to 90 °C. Combined with the Tm that was found to be 78.5 ± 0.5 °C at pH 8.0, TfSOD can be defined as a thermostable enzyme. Moreover, the crystal structure of TfSOD was determined and refined to 1.25 Å resolution. With electron paramagnetic resonance spectroscopy, it was confirmed that iron is the metal co-factor of TfSOD. The cell potential (Em) for the TfSOD-Fe3+/TfSOD-Fe2+ redox couple was determined to be 287 mV.Graphical abstract

Combination of sequencing batch reactor activated sludge process with sludge lysis using thermophilic bacterial community for minimizing excess sludge

Sludge reduction is a major challenge in biological wastewater treatment. Hydrolytic enzymes secreted by thermophilic bacteria can lyse sludge and thus achieve sludge reduction, and the indigenous thermophilic community in sludge can lyse sludge more effectively. In this study, the feasibility of combining a sludge lysis reactor based on thermophilic bacteria community (LTBC reactor, 75 °C) with a conventional sequencing batch activated sludge reactor (SBR) for sludge reduction (i.e., LTBC-SBR process) was systematically investigated first time. The effect of lysed sludge returning to the biochemical tank on pollutant removal efficiency, sludge flocculation, sludge settling, and microbial community and function of the LTBC-SBR process was studied. In the LTBC1-SBR process, a sludge growth rate of 0.71 g TSS/day was observed when the lysed sludge reflux ratio (LRR) was 1, and the sludge generation was reduced by 81.5% compared to the conventional SBR reactor. In the LTBC1-SBR process, the removal efficiencies of chemical oxygen demand and total nitrogen were 94.0% and 80.5%, respectively. There was no significant difference in the sludge volume index from the SBR to the LTBC1-SBR stage, however, the effluent suspended solids concentration increased from 35.2 ± 2.1 mg/L to 80.1 ± 5.3 mg/L. This was attributed to the reflux of sludge lysate. In addition, the changes in extracellular polymers content and composition resulted in poor sludge flocculation performance. Heterotrophic bacteria associated with Actinobacteria and Patescibacteria enriched in LTBC1-SBR with relative abundance of 28.51 ± 1.25% and 20.01 ± 1.21%, respectively, which decomposed the macromolecules in the refluxed lysed sludge and contributed to the sludge reduction. Furthermore, due to the inhibition of nitrite-oxidizing bacteria, the nitrite concentration in the effluent of the LTBC1-SBR system reached 4.7 ± 1.1 mg/L, and part of the denitrification process was achieved by short-cut nitrification and simultaneous denitrification. These results indicate that in-situ sludge reduction technology based on lyse sludge lysing by thermophilic community has considerable potential to be widely used in wastewater treatment.

Pemanfaatan Bakteri Termofilik Penghasil Amilase Pada Berbagai Pati

Indonesia is a country that produces a lot of starchy materials such as cassava, sago, corn and others. Indonesia has the potential to develop an enzyme-producing industry, especially amylase. The utilization of starchy materials which so far has not been maximized and is still limited to conventional methods can be increased by enzymatic development processes. This will increase its economic value and at the same time increase state income. One of the enzymes that is currently very widely used in the food and beverage industry is amylase. Nin Suharti and Geminsyah Putra 2021 have succeeded in isolating and identifying thermophilic bacteria from the sidedust hot springs of North Sumatra. The advantage of thermophilic bacteria is that they can produce enzymes that are resistant to high temperatures / thermostable enzymes. These enzymes are able to survive and are active at high temperatures. Such properties are highly needed by enzyme-based industries. The use of enzymes that can withstand high temperatures in the field of biotechnology can reduce operating costs and increase reaction speed, this study uses various types of starch including potatoes, cassava and corn as suitable and easily available carbon sources for the production of amylase enzymes. Bacteria in the media with potato starch added can produce the highest amylase enzyme from the three treatments of 65 U/L. For bacteria added with corn starch, the result was 50 U/L of amylase enzyme. Meanwhile, the lowest of the three treatments was media with added sweet potato starch with a content of 39 U/L. The isolates generally had a round shape, jagged edges, grooved, and smooth, white colonies, microscopic bacilli-shaped, endospores and motile, positive for catalase, gelatin and citrate tests. With Gram staining, it generally shows a positive Gram reaction.

Similarities of Geobacillus bacteria based on their profiles of antimicrobial susceptibility in milk samples

The genus Geobacillus is composed of thermophilic bacteria that exhibit diverse biotechnological potentialities. Specifically, Geobacillus stearothermophilus is included as a test bacterium in commercial microbiological inhibition methods, although it exhibits limited sensitivity to aminoglycosides, macrolides, and quinolones. Therefore, this article evaluates the antibiotic susceptibility profiles of five test bacteria (G. stearothermophilus subsp. calidolactis C953, Geobacillus thermocatenulatus LMG 19007, Geobacillus thermoleovorans LMG 9823, Geobacillus kaustophilus DSM 7263 and Geobacillus vulcani 13174). For that purpose, the minimum inhibitory concentrations (MICs) of 21 antibiotics were determined in milk samples for five test bacteria using the radial diffusion microbiological inhibition method. Subsequently, the similarities between bacteria and antibiotics were analyzed using cluster analysis. The dendrogram of this multivariate analysis shows an association between a group formed by G. thermocatenulatus and G. stearothermophilus and another by G. thermoleovorans, G. kaustophilus and G. vulcani. Finally, future microbiological methods could be developed in microtiter plates using G. thermocatenulatus as test bacterium, as it exhibits similar sensitivities to G. stearothermophilus. Conversely, G. vulcani, G. thermoleovorans and G. kaustophilus show higher MICs than G. thermocatenulatus.

Identifying the role of array electrodes in improving the compost quality of food waste during electric field-assisted aerobic composting

Low composting temperature and long maturation periods are two major problems during food waste composting. In this study, a novel array-based electric field-assisted aerobic composting (Pin-EAC) process was tested on food waste compost. Pin-EAC increase the composting temperature to 69.3 °C, and improved the germination index by 15%. The Pin-EAC took at least 40% less time to reach the standard compost maturity. The fluorescent spectroscopy results showed that Pin-EAC could increase humic acid and fulvic acid by 33% and 37%, respectively. Pin-EAC could increase the diversity of thermophilic bacteria during composting. The co-occurrence network shown that Pin-EAC are more closely related to oxygen and temperature. This work has initially shown that the use of an electric field could improve food waste composting quality, suggesting that the Pin-EAC process is an effective strategy for high-water and high-oil organic solid waste aerobic composting.

Comparison of Structural Features of CRISPR-Cas Systems in Thermophilic Bacteria.

The clustered regularly interspaced short palindromic repeat (CRISPR) is an adaptive immune system that defends most archaea and many bacteria from foreign DNA, such as phages, viruses, and plasmids. The link between the CRISPR-Cas system and the optimum growth temperature of thermophilic bacteria remains unclear. To investigate the relationship between the structural characteristics, diversity, and distribution properties of the CRISPR-Cas system and the optimum growth temperature in thermophilic bacteria, genomes of 61 species of thermophilic bacteria with complete genome sequences were downloaded from GenBank in this study. We used CRISPRFinder to extensively study CRISPR structures and CRISPR-associated genes (cas) from thermophilic bacteria. We statistically analyzed the association between the CRISPR-Cas system and the optimum growth temperature of thermophilic bacteria. The results revealed that 59 strains of 61 thermophilic bacteria had at least one CRISPR locus, accounting for 96.72% of the total. Additionally, a total of 362 CRISPR loci, 209 entirely distinct repetitive sequences, 131 cas genes, and 7744 spacer sequences were discovered. The average number of CRISPR loci and the average minimum free energy (MFE) of the RNA secondary structure of repeat sequences were positively correlated with temperature whereas the average length of CRISPR loci and the average number of spacers were negatively correlated. The temperature did not affect the average number of CRISPR loci, the average length of repeats, or the guanine-cytosine (GC) content of repeats. The average number of CRISPR loci, the average length of the repeats, and the GC content of the repeats did not reflect temperature dependence. This study may provide a new basis for the study of the thermophilic bacterial adaptation mechanisms of thermophilic bacteria.

Syntrophic entanglements for propionate and acetate oxidation under thermophilic and high-ammonia conditions

Propionate is a key intermediate in anaerobic digestion processes and often accumulates in association with perturbations, such as elevated levels of ammonia. Under such conditions, syntrophic ammonia-tolerant microorganisms play a key role in propionate degradation. Despite their importance, little is known about these syntrophic microorganisms and their cross-species interactions. Here, we present metagenomes and metatranscriptomic data for novel thermophilic and ammonia-tolerant syntrophic bacteria and the partner methanogens enriched in propionate-fed reactors. A metagenome for a novel bacterium for which we propose the provisional name ‘Candidatus Thermosyntrophopropionicum ammoniitolerans’ was recovered, together with mapping of its highly expressed methylmalonyl-CoA pathway for syntrophic propionate degradation. Acetate was degraded by a novel thermophilic syntrophic acetate-oxidising candidate bacterium. Electron removal associated with syntrophic propionate and acetate oxidation was mediated by the hydrogen/formate-utilising methanogens Methanoculleus sp. and Methanothermobacter sp., with the latter observed to be critical for efficient propionate degradation. Similar dependence on Methanothermobacter was not seen for acetate degradation. Expression-based analyses indicated use of both H2 and formate for electron transfer, including cross-species reciprocation with sulphuric compounds and microbial nanotube-mediated interspecies interactions. Batch cultivation demonstrated degradation rates of up to 0.16 g propionate L−1 day−1 at hydrogen partial pressure 4–30 Pa and available energy was around −20 mol−1 propionate. These observations outline the multiple syntrophic interactions required for propionate oxidation and represent a first step in increasing knowledge of acid accumulation in high-ammonia biogas production systems.

Isolation and screening of thermophillic bacteria and its subsequent evaluation for lipases production

Isolation and screening of thermophillic bacteria and its subsequent evaluation for lipases production

Customized multiple sequence alignment as an effective strategy to improve performance of Taq DNA polymerase.

Engineering Taq DNA polymerase (TaqPol) for improved activity, stability and sensitivity was critical for its wide applications. Multiple sequence alignment (MSA) has been widely used in engineering enzymes for improved properties. Here, we first designed TaqPol mutations based on MSA of 2756 sequences from both thermophilic and non-thermophilic organisms. Two double mutations were generated including a variant H676F/R677G showing a decrease in both activity and stability, and a variant Y686R/E687K showing an improved activity, but a decreased stability. Mutations targeted on coevolutionary residues of Arg677 and Tyr686 were then applied to rescue stability or activity loss of the double mutants, which achieved a partial success. Sequence analysis revealed that the two mutations are abundant in non-thermophilic sequences but not in thermophilic homologues. Then, a small-scale MSA containing sequences from only thermophilic organisms was applied to predict 13 single variants and two of them, E507Q and E734N showed a simultaneous increase in both stability and activity, even in sensitivity. A customized MSA was hence more effective in engineering a thermophilic enzyme and could be used in engineering other enzymes. Molecular dynamics simulations revealed the impact of mutations on the protein dynamics and interactions between TaqPol and substrates. KEY POINTS: • The pool of sequence for alignment is critical to engineering Taq DNA polymerase. • The variants with low properties can be rescued by mutations in coevolving network. • Improving binding with DNA can improve DNA polymerase stability and activity.

Simple Assessing of Calcification Catalyzed by Thermophilic Bacteria

Several bacterial species have been found to involve the biomineralization phenomenon promoted by them. The formation of calcium carbonate mineral (calcification) is most abundant phenomena as biomineralization by bacteria. Here we introduce the simple assessing method of calcification catalyzed by thermophilic bacteria. To obtain the fresh biomass of thermophilic bacterium, the thermophilic bacteria was cultured on conventional nutrient agar medium at 60˚C. Fresh biomass of bacteria is simply placed on calcite promoting hydrogel surface, and incubated at 60˚C. After incubation for 24 to 72 h, a number of single crystals can be found in the biomass. This method provides simple assessing for screening of calcification upon thermophilic bacteria.

Production and evaluation of a mutant galactose-utilizing strain of Streptococcus thermophilus for application in milk fermentation

Streptococcus thermophilus is a thermophilic gram-positive bacterium used in the production of fermented milk. Most S. thermophilus strains originating from milk can only use the glucose moiety of lactose as a carbon source, while the galactose moiety is secreted into the fermenting milk. In this study, one galactose-positive mutant (S. thermophilus IMAU80285Y) was produced by treating S. thermophilus IMAU80285 with the chemical mutagen, N-methyl-N′-nitro-N-nitrosoguanidine (NTG), and fermentation characteristics were evaluated. β-Galactosidase activity was higher in the mutant strain while glucokinase and pyruvate kinase activities were significantly decreased compared with the wild type. Sequencing revealed that S. thermophilus IMAU80285Y had mutations in the β-galactosidase, glucokinase and pyruvate genes. High performance liquid chromatography (HPLC) analysis demonstrated that accumulation of galactose in milk fermented by the mutant S. thermophilus IMAU80285Y was reduced by 55.4%, compared with the wild strain. Our results indicate that mutation enabled rerouting of the galactose metabolism pathways and provides new insights into using chemical mutagenesis to select strains with excellent characteristics for dairy application.

Moderately Thermophilic Bacteria from Jordanian Hot Springs as Possible Sources of Thermostable Enzymes and Leukemia Cytotoxic Agents

Moderately Thermophilic Bacteria from Jordanian Hot Springs as Possible Sources of Thermostable Enzymes and Leukemia Cytotoxic Agents

Characterization of Thermostable Cellulase from Bacillus licheniformis PANG L Isolated from the Himalayan Soil.

This study aimed to isolate, purify, and characterize a potential thermophilic cellulase-producing bacterium from the Himalayan soil. Eleven thermophilic bacteria were isolated, and the strain PANG L was found to be the most potent cellulolytic producer. Morphological, physiological, biochemical, and molecular characterization identified PANG L as Bacillus licheniformis. This is the first study on the isolation of thermostable cellulase-producing Bacillus licheniformis from the Himalayan soil. This bacterium was processed for the production of cellulase enzyme. The optimum conditions for cellulase production were achieved at 45°C after 48 h of incubation at pH 6.5 in media-containing carboxymethyl cellulose (CMC) and yeast extract as carbon and nitrogen sources, respectively, in a thermo-shaker at 100 rpm. The enzyme was partially purified by 80% ammonium sulphate precipitation followed by dialysis, resulting in a 1.52-fold purification. The optimal activity of partially purified cellulase was observed at a temperature of 60°C and pH 5. The cellulase enzyme was stable within the pH ranges of 3-5 and retained 67% of activity even at 55°C. Cellulase activity was found to be enhanced in the presence of metal ions such as Cd2+, Pb2+, and Ba2+. The enzyme showed the highest activity when CMC was used as a substrate, followed by cellobiose. The Km and Vmax values of the enzyme were 1.8 mg/ml and 10.92 μg/ml/min, respectively. The cellulase enzyme obtained from Bacillus licheniformis PANG L had suitable catalytic properties for use in industrial applications.

Exploring Anaerobic Digestion from Mesophilic to Thermophilic Temperatures—Operational and Microbial Aspects

Digesters at water resource recovery facilities (WRRFs) operating at different temperatures within the mesophilic and thermophilic temperature range is a flexibilization concept to contribute to heat management. Four 25 L digesters were fed with sewage sludge from a municipal WRRF and were operated at 37, 43, 47 and 53 °C, respectively, to describe changes in the overall process performance and the microbiota. Specific methane yield and COD degradation rates were the highest at 47 °C, only being up to 7% higher compared with at 37 °C. The increase in pH and concentrations of NH4-N and PO4-P above 43 °C were statistically significant. The effect on the microbial community was strong, indicating both a constant specialization towards thermophilic organisms as well as a change from acetoclastic to hydrogenotrophic/methylotrophic methanogenesis. The influence of temperature on process-engineering and physicochemical aspects was rather small compared with the changes in the microbiota.

Biooxidation of hydrogen sulfide to sulfur by moderate thermophilic acidophilic bacteria.

The copper industry utilizes significant amounts of sulfuric acid in its processes, generating sulfate as waste. While sulfate-reducing bacteria can remove sulfate, it produces hydrogen sulfide (H2S) as a byproduct. This study examined the capability of a consortium consisting of Sulfobacillus thermosulfidooxidans and Sulfobacillus acidophilus to partially oxidize H2S to S° at a temperature of 45°C. A fixed-bed bioreactor, with glass rings as support material and sodium thiosulfate as a model electron donor, was inoculated with the consortium. Formation of biofilms was crucial to maintain the bioreactor's steady state, despite high flow rates. Afterward, the electron donor was changed to H2S. When the bioreactor was operated continuously and with high aeration, H2S was fully oxidized to SO42-. However, under conditions of low aeration and at a concentration of 0.26g/L of H2S, the consortium was able to oxidize H2S to S° with a 13% yield. S° was discovered attached to the glass rings and jarosite. The results indicate that the consortium could oxidize H2S to S° with a 13% yield under low aeration and at a concentration of 0.26g/L of H2S. The findings highlight the capability of a Sulfobacillus consortium to convert H2S into S°, providing a potential solution for addressing environmental and safety issues associated with sulfate waste generated by the mining industry.

Exploration of Thermophilic Bacteria: Systematic Literature Review

The selection of enzymes derived from microbes has several advantages compared to those derived from animals and plants. One of the advantages is that the growth of enzymes will occur more quickly in bacteria because the cells rapidly undergo division and cell production will be easier to increase if needed in large quantities and the time needed for enzyme production will be shorter. Thermophilic microorganisms are known as bacteria that are tolerant of high temperatures, but on the other hand these bacteria are also able to survive at these temperatures. Where the purpose of research is to explain exploration of thermophilic bacteria. A review is conducted on the state-of-the-art methods using the preferred reporting items for reviews and meta-analyses (PRISMA) guideline. Bacteria have important economic value in human life. Knowledge in this branch of science is useful in medicine, hygiene, food science and nutrition, agriculture, and industry. Based on the results of macroscopic, microscopic and biochemical characterization, it was found that many bacteria with various types recording to their habitat or place of development, exploraton of thermophilic bacteria was carried out so that they could be useful for the industrial world.