Thermophilic microorganisms involved in the nitrogen cycle in thermal environments: Advances and prospects.

Thermophilic microorganisms mediated significant element cycles and material conversion in the early Earth as well as mediating current thermal environments. Over the past few years, versatile microbial communities that drive the nitrogen cycle have been identified in thermal environments. Understanding the microbial-mediated nitrogen cycling processes in these thermal environments has important implications for the cultivation and application of thermal environment microorganisms as well as for exploring the global nitrogen cycle. This work provides a comprehensive review of different thermophilic nitrogen-cycling microorganisms and processes, which are described in detail according to several categories, including nitrogen fixation, nitrification, denitrification, anaerobic ammonium oxidation, and dissimilatory nitrate reduction to ammonium. In particular, we assess the environmental significance and potential applications of thermophilic nitrogen-cycling microorganisms, and highlight knowledge gaps and future research opportunities.

Exploring Extremophiles from Bulgaria: Biodiversity, Biopolymer Synthesis, Functional Properties, Applications.

Bulgaria stands out as a country rich in diverse extreme environments, boasting a remarkable abundance of mineral hot waters, which positions it as the second-largest source of such natural resources in Europe. Notably, several thermal and coastal solar salterns within its territory serve as thriving habitats for thermophilic and halophilic microorganisms, which offer promising bioactive compounds, including exopolysaccharides (EPSs). Multiple thermophilic EPS producers were isolated, along with a selection from several saltern environments, revealing an impressive taxonomic and bacterial diversity. Four isolates from three different thermophilic species, Geobacillus tepidamans V264, Aeribacillus pallidus 418, Brevibacillus thermoruber 423, and Brevibacillus thermoruber 438, along with the halophilic strain Chromohalobacter canadensis 28, emerged as promising candidates for further exploration. Optimization of cultivation media and conditions was conducted for each EPS producer. Additionally, investigations into the influence of aeration and stirring in laboratory bioreactors provided valuable insights into growth dynamics and polymer synthesis. The synthesized biopolymers showed excellent emulsifying properties, emulsion stability, and synergistic interaction with other hydrocolloids. Demonstrated biological activities and functional properties pave the way for potential future applications in diverse fields, with particular emphasis on cosmetics and medicine. The remarkable versatility and efficacy of biopolymers offer opportunities for innovation and development in different industrial sectors.

Insight into the effect of nitrogen-rich substrates on the community structure and the co-occurrence network of thermophiles during lignocellulose-based composting.

Thermophilic microorganisms play vital roles in the composting process. To elucidate how raw materials affect thermophilic microbial community composition and their interactions, the succession of thermophilic bacterial and fungal communities were monitored in reed straw co-composting with four common nitrogen-rich substrates. The results of high-throughput sequencing showed that raw materials and composting process significantly changed bacterial and fungal community composition. Firmicutes and Actinobacteria drove the assembly of bacterial communities, while Ascomycetes drove the assembly of fungal communities. Network analysis indicated that during the composting process, the addition of nitrogen-rich sources abundant in easily degradable substances promoted the complexity of thermophilic microbial network. Moreover, microorganisms mainly exhibited synergistic effects, and inter-kingdom competition was more intense than intra-kingdom competition. Notably, rare species play essential roles in maintaining the network construction. Our findings provided novel insights into thermophilic microbial community assembly and their co-occurrence networks during the composting process.

A perspective on biotechnological applications of thermophilic microalgae and cyanobacteria.

The importance of expanding our knowledge on microorganisms derived from extreme environments stems from the development of novel and sustainable technologies for our health, food, and environment. Microalgae and cyanobacteria represent a group of diverse microorganisms that inhabit a wide range of environments, are capable of oxygenic photosynthesis, and form a thick microbial mat even at extreme environments. Studies of thermophilic microorganisms have shown a considerable biotechnological potential due to their optimum growth and metabolisms at high temperatures (≥50 °C), which is supported by their thermostable enzymes. Microalgal and cyanobacterial communities present in high-temperature ecosystems account for a large part of the total ecosystem biomass and productivity, and can be exploited to generate several value-added products of agricultural, pharmaceutical, nutraceutical, and industrial relevance. This review provides an overview on the current status of biotechnological applications of thermophilic microalgae and cyanobacteria, with an outlook on the challenges and future prospects.

Occurrence of Thermophilic Microorganisms in Different Full Scale Biogas Plants.

BACKGROUND: In recent years, various substrates have been tested to increase the sustainable production of biomethane. The effect of these substrates on methanogenesis has been investigated mainly in small volume fermenters and were, for the most part, focused on studying the diversity of mesophilic microorganisms. However, studies of thermophilic communities in large scale operating mesophilic biogas plants do not yet exist. METHODS: Microbiological, biochemical, biophysical methods, and statistical analysis were used to track thermophilic communities in mesophilic anaerobic digesters. RESULTS: The diversity of the main thermophile genera in eight biogas plants located in the Czech Republic using different input substrates was investigated. In total, 19 thermophilic genera were detected after 16S rRNA gene sequencing. The highest percentage (40.8%) of thermophiles was found in the Modrice biogas plant where the input substrate was primary sludge and biological sludge (50/50, w/w %). The smallest percentage (1.87%) of thermophiles was found in the Cejc biogas plant with the input substrate being maize silage and liquid pig manure (80/20, w/w %). CONCLUSIONS: The composition of the anaerobic consortia in anaerobic digesters is an important factor for the biogas plant operator. The present study can help characterizing the impact of input feeds on the composition of microbial communities in these plants.

Identificação de micro-organismos presentes em sistemas de biolixiviação de cobre a 35 e 50°C / Identifying microorganisms related to copper bioleaching at 35 and 50°C

RESUMO A biolixiviação de minérios de baixo teor e com elevado conteúdo de impurezas tem se mostrado alternativa importante para o aproveitamento destes, uma vez que a recuperação do metal por métodos pirometalúrgicos convencionais mostra-se economicamente inviável. A identificação e quantificação dos micro-organismos capazes de promover a biolixiviação mostram-se estratégicas para alcançar bons rendimentos no controle do processo e na recuperação de metais. Nesse sentido, as técnicas de biologia molecular são as ferramentas mais utilizadas para tal propósito. Este trabalho, utilizando técnicas de reação em cadeia da polimerase (PCR), polimorfismos de comprimento dos fragmentos de restrição (RFLP) e reação em cadeia da polimerase seguida de eletroforese em gel com gradiente desnaturante (PCR-DGGE), mostrou que a diversidade nas colunas de biolixiviação de cobre estudadas é baixa e que a temperatura é importante na manutenção de determinadas espécies, havendo predominância de Acidithiobacillus ferroxidans a 35°C e de Sulfobacillus thermosulfidooxidans a 50°C.

ABSTRACT Bioleaching is an alternative to pyrometallurgy for the production of metals from low-grade ores containing high level of impurities, once that live pyrometallurgical methods are economically unfeasible. The quantification and identification of those microorganisms related to bioleaching is an important strategy for process control and thus metal recovery. In this regard, molecular biology is one of the main techniques utilized for such objective. This study applied PCR, RFLP and PCR-DGGE techniques to show that the microbial diversity in copper bioleaching columns under investigation is low and the temperature is important to define the species found, with predominance of Acidithiobacillus ferroxidans, at 35°C and Sulfobacillus thermosulfidooxidans at 50°C.

High-pressure thermophilic electromethanogenic system producing methane at 5 MPa, 55°C.

Toward applications of bio-electrochemical systems in industrial processes and extreme environments, electromethanogenesis under high-pressure conditions was examined. Stainless-steel single-chamber reactors specifically designed to examine bio-electrochemical reactions under pressurized conditions were inoculated with thermophilic microorganisms originated from an oilfield formation water. The reactors were incubated at 5 MPa, 55°C in fed-batch operational mode with an applied voltage of 0.7 V. In the first few fed-batch cycles, hydrogen was mainly produced. After the third cycle, however, the reactors produced only methane simultaneously with current generation. The methane-production rate of the reactors showed an applied-voltage dependence and increased from 34.9 to 168.4 mmol m-2 day-1 with an increase in the applied voltage from 0.4 to 0.9 V. The efficiency of capturing electrons in the produced methane on average exceeded 70% with the applied voltage of 0.4 V or higher. Cyclic voltammetry further confirmed abilities of the bioelectrodes to catalyze electrochemical reactions at 5 MPa. Performance of the electromethanogenesis system was not altered under lower pressure conditions (1.2 and 2.5 MPa). An exoelectrogenic bacterium affiliated with the genus Thermincola and a methanogen belonging to the genus Methanothermobacter were detected as the dominant species in the bioanode and biocathode microbiotas, respectively. Thus, our results indicated that electromethanogenesis systems could be developed and operated under highly-pressurized conditions, suggesting that applications of the bio-electrochemical system in high-pressure environments (including high-temperature subsurface reservoirs) can be technically feasible.

Thermostable Xanthine Oxidase Activity from Bacillus pumilus RL-2d Isolated from Manikaran Thermal Spring: Production and Characterization.

Xanthine oxidase is an important enzyme of purine metabolism that catalyzes the hydroxylation of hypoxanthine to xanthine and then xanthine to uric acid. A thermostable xanthine oxidase is being reported from a thermophilic organism RL-2d isolated from the Manikaran (Kullu) hot spring of Himachal Pradesh (India). Based on the morphology, physiological tests, and 16S rDNA gene sequence, RL-2d was identified as Bacillus pumilus. Optimization of physiochemical parameters resulted into 4.1-fold increase in the xanthine oxidase activity from 0.051 U/mg dcw (dry cell weight) to 0.209 U/mg dcw. The xanthine oxidase of B. pumilus RL-2d has exhibited very good thermostability and its t1/2 at 70 and 80 °C were 5 and 1 h, respectively. Activity of this enzyme was strongly inhibited by Hg(2+), Ag(+) and allopurinol. The investigation showed that B. pumilus RL-2d exhibited highest xanthine oxidase activity and remarkable thermostability among the other xanthine oxidases reported so far.

A Cascade of Thermophilic Enzymes As an Approach to the Synthesis of Modified Nucleotides.

We propose a new approach for the synthesis of biologically important nucleotides which includes a multi-enzymatic cascade conversion of D-pentoses into purine nucleotides. The approach exploits nucleic acid exchange enzymes from thermophilic microorganisms: ribokinase, phosphoribosylpyrophosphate synthetase, and adenine phosphoribosyltransferase. We cloned the ribokinase gene from Thermus sp. 2.9, as well as two different genes of phosphoribosylpyrophosphate synthetase (PRPP-synthetase) and the adenine phosphoribosyltransferase (APR-transferase) gene from Thermus thermophilus HB27 into the expression vectors, generated high-yield E. coli producer strains, developed methods for the purification of the enzymes, and investigated enzyme substrate specificity. The enzymes were used for the conversion of D-pentoses into 5-phosphates that were further converted into 5-phospho-α-D-pentofuranose 1-pyrophosphates by means of ribokinase and PRPP-synthetases. Target nucleotides were obtained through the condensation of the pyrophosphates with adenine and its derivatives in a reaction catalyzed by APR-transferase. 2-Chloro- and 2-fluoroadenosine monophosphates were synthesized from D-ribose and appropriate heterobases in one pot using a system of thermophilic enzymes in the presence of ATP, ribokinase, PRPP-synthetase, and APR-transferase.

Insight into thermophiles and their wide-spectrum applications.

The deconstruction of biomass is a pivotal process for the manufacture of target products using microbial cells and their enzymes. But the enzymes that possess a significant role in the breakdown of biomass remain relatively unexplored. Thermophilic microorganisms are of special interest as a source of novel thermostable enzymes. Many thermophilic microorganisms possess properties suitable for biotechnological and commercial use. There is, indeed, a considerable demand for a new generation of stable enzymes that are able to withstand severe conditions in industrial processes by replacing or supplementing traditional chemical processes. This manuscript reviews the pertinent role of thermophilic microorganisms as a source for production of thermostable enzymes, factors afftecting them, recent patents on thermophiles and moreso their wide spectrum applications for commercial and biotechnological use.

Thermophilic lignocellulose deconstruction.

Thermophilic microorganisms are attractive candidates for conversion of lignocellulose to biofuels because they produce robust, effective, carbohydrate-degrading enzymes and survive under harsh bioprocessing conditions that reflect their natural biotopes. However, no naturally occurring thermophile is known that can convert plant biomass into a liquid biofuel at rates, yields and titers that meet current bioprocessing and economic targets. Meeting those targets requires either metabolically engineering solventogenic thermophiles with additional biomass-deconstruction enzymes or engineering plant biomass degraders to produce a liquid biofuel. Thermostable enzymes from microorganisms isolated from diverse environments can serve as genetic reservoirs for both efforts. Because of the sheer number of enzymes that are required to hydrolyze plant biomass to fermentable oligosaccharides, the latter strategy appears to be the preferred route and thus has received the most attention to date. Thermophilic plant biomass degraders fall into one of two categories: cellulosomal (i.e. multienzyme complexes) and noncellulosomal (i.e. 'free' enzyme systems). Plant-biomass-deconstructing thermophilic bacteria from the genera Clostridium (cellulosomal) and Caldicellulosiruptor (noncellulosomal), which have potential as metabolic engineering platforms for producing biofuels, are compared and contrasted from a systems biology perspective.

04-1 Fangotherapy, a body-friendly treatment to use hot spring water for health promotion / 日本温泉気候物理医学会雑誌

Fangotherapy is one of four medical treatments used under a medical doctor’s supervision at a hot spring. This treatment is conducted in Europe, especially in Italy, using peloids maturated with natural hot spring water. The maturated peloids have factors of concentrated hot spring and biological extract -glycolipids- from thermophilic algae.   There are many kinds of hot springs in Japan, but they are almost always used for “taking a bath” only. Our research focused on Fango found in Abano Italy, and modified into “Japanese style Fango” made with maturated peloids by hot spring water. Methods: The original Japanese Fango, which we made in a hot spring in Japan, has been named Biofango®. The original and first Biofango® was made from the Sanrakuen Hotel’s hot spring water in Toyama, and treated in the hotel as in the Abano style. The benefits of Biofango® were checked using the following methods; 1.　Hyperthermia and some medical effects were checked under treatment with maturated peloids, and the effects were compared with hot spring water only and with boiled tap water. 2.　The double-blind method was used comparing Biofango® (True Fango) and imitation Fango. Two kinds of Fango were made using either hot spring water or hot tap water, and some medical effects were checked. After treatment of Fangotherapy, a medical questionnaire was provided for each test subject. Results: Fango (Biofango®) is the best treatment among three bathing methods for keeping normal responses of blood flow and blood pressure at the thigh, and for keeping good thermal effects on the body, especially for the back of the body after 50 min.   The results of the double-blind method, deep body temperature increase and diastolic blood pressure decrease in True Fango showed a significant difference. Pulse increase and SIV decrease in True Fango shows a reduced load on the vessel, while still showing a high thermal effect. Functions of the body showed an improved friend in True Fango, according to the questionnaire survey. Conclusion: Traditional use of hot spring water in Japan was hot spring bathing in the mainstream. But, according to these results, Fango is the best method for a body-friendly treatment by hot spring water. Further, the questionnaire about body functions after Fango treatment shows that the hot spring Fango (True Fango) is more effective than hot tap water Fango (imitation Fango).   In Italy, glycolipids from thermophilic algae are an important factor for reducing inflammation by Fango therapy. Growth of thermophilic algae also has been confirmed in Fango maturation in Japan, and was cultured in the laboratory. We also found such glycolipids from Japanese algae, and have evidence, in situ, of the glycolipids remaining between particles of peloids. In the future, Biofango® should have a useful biological factor similar to Abano Fango for treatment.

04-1Fangotherapy, a body-friendly treatment to use hot spring water for health promotion / 日本温泉気候物理医学会雑誌

Fangotherapy is one of four medical treatments used under a medical doctor’s supervision at a hot spring. This treatment is conducted in Europe, especially in Italy, using peloids maturated with natural hot spring water. The maturated peloids have factors of concentrated hot spring and biological extract -glycolipids- from thermophilic algae. <BR>  There are many kinds of hot springs in Japan, but they are almost always used for “taking a bath” only. Our research focused on Fango found in Abano Italy, and modified into “Japanese style Fango” made with maturated peloids by hot spring water. <BR><b>Methods: </b>The original Japanese Fango, which we made in a hot spring in Japan, has been named Biofango^®. The original and first Biofango^® was made from the Sanrakuen Hotel’s hot spring water in Toyama, and treated in the hotel as in the Abano style. The benefits of Biofango^® were checked using the following methods;<BR>1.　Hyperthermia and some medical effects were checked under treatment with maturated peloids, and the effects were compared with hot spring water only and with boiled tap water. <BR>2.　The double-blind method was used comparing Biofango^® (True Fango) and imitation Fango. Two kinds of Fango were made using either hot spring water or hot tap water, and some medical effects were checked. After treatment of Fangotherapy, a medical questionnaire was provided for each test subject.<BR><b>Results: </b>Fango (Biofango^®) is the best treatment among three bathing methods for keeping normal responses of blood flow and blood pressure at the thigh, and for keeping good thermal effects on the body, especially for the back of the body after 50 min. <BR>  The results of the double-blind method, deep body temperature increase and diastolic blood pressure decrease in True Fango showed a significant difference. Pulse increase and SIV decrease in True Fango shows a reduced load on the vessel, while still showing a high thermal effect. Functions of the body showed an improved friend in True Fango, according to the questionnaire survey.<BR><b>Conclusion: </b>Traditional use of hot spring water in Japan was hot spring bathing in the mainstream. But, according to these results, Fango is the best method for a body-friendly treatment by hot spring water. Further, the questionnaire about body functions after Fango treatment shows that the hot spring Fango (True Fango) is more effective than hot tap water Fango (imitation Fango).<BR>  In Italy, glycolipids from thermophilic algae are an important factor for reducing inflammation by Fango therapy. Growth of thermophilic algae also has been confirmed in Fango maturation in Japan, and was cultured in the laboratory. We also found such glycolipids from Japanese algae, and have evidence, <i>in situ</i>, of the glycolipids remaining between particles of peloids. In the future, Biofango^® should have a useful biological factor similar to Abano Fango for treatment.