Hotspot Wizard-informed engineering of a hyperthermophilic ß-glucosidase for enhanced enzyme activity at low temperatures.

Hyperthermophilic enzymes serve as an important source of industrial enzymes due to their high thermostability. Unfortunately, most hyperthermophilic enzymes suffer from reduced activity at low temperatures (e.g., ambient temperature), limiting their applicability. In addition, evolving hyperthermophilic enzymes to increase low temperature activity without compromising other desired properties is generally difficult. In the current study, a variant of ß-glucosidase from Pyrococcus furiosus (PfBGL) was engineered to enhance enzyme activity at low temperatures through the construction of a saturation mutagenesis library guided by the HotSpot Wizard analysis, followed by its screening for activity and thermostability. From this library construction and screening, one PfBGL mutant, PfBGL-A4 containing Q214S/A264S/F344I mutations, showed an over twofold increase in ß-glucosidase activity at 25 and 50°C compared to the wild type, without compromising high-temperature activity, thermostability and substrate specificity. Our experimental and computational characterizations suggest that the findings with PfBGL-A4 may be due to the elevation of local conformational flexibility around the active site, while slightly compacting the global protein structure. This study showcases the potential of HotSpot Wizard-informed engineering of hyperthermophilic enzymes and underscores the interplays among temperature, enzyme activity, and conformational flexibility in these enzymes.

A filamentous archaeal virus is enveloped inside the cell and released through pyramidal portals.

The majority of viruses infecting hyperthermophilic archaea display unique virion architectures and are evolutionarily unrelated to viruses of bacteria and eukaryotes. The lack of relationships to other known viruses suggests that the mechanisms of virus-host interaction in Archaea are also likely to be distinct. To gain insights into archaeal virus-host interactions, we studied the life cycle of the enveloped, ∼2-µm-long Sulfolobus islandicus filamentous virus (SIFV), a member of the family Lipothrixviridae infecting a hyperthermophilic and acidophilic archaeon Saccharolobus islandicus LAL14/1. Using dual-axis electron tomography and convolutional neural network analysis, we characterize the life cycle of SIFV and show that the virions, which are nearly two times longer than the host cell diameter, are assembled in the cell cytoplasm, forming twisted virion bundles organized on a nonperfect hexagonal lattice. Remarkably, our results indicate that envelopment of the helical nucleocapsids takes place inside the cell rather than by budding as in the case of most other known enveloped viruses. The mature virions are released from the cell through large (up to 220 nm in diameter), six-sided pyramidal portals, which are built from multiple copies of a single 89-amino-acid-long viral protein gp43. The overexpression of this protein in Escherichia coli leads to pyramid formation in the bacterial membrane. Collectively, our results provide insights into the assembly and release of enveloped filamentous viruses and illuminate the evolution of virus-host interactions in Archaea.

Acceleration of the biodegradation of cationic polyacrylamide by the coupling effect of thermophilic microorganisms and high temperature in hyperthermophilic composting.

As a flocculant of sewage sludge, cationic polyacrylamide (CPAM) enters the environment with sludge and exists for a long time, posing serious threats to the environment. Due to the environmental friendliness and high efficiency in the process of organic solid waste treatment, hyperthermophilic composting (HTC) has received increasing attention. However, it is still unclear whether the HTC process can effectively remove CPAM from sludge. In this study, the effects of HTC and conventional thermophilic composting (CTC) on CPAM in sludge were compared and analyzed. At the end of HTC and CTC, the concentrations of CPAM were 278.96 mg kg-1 and 533.89 mg kg-1, respectively, and the removal rates were 72.17% and 46.61%, respectively. The coupling effect of thermophilic microorganisms and high temperature improved the efficiency of HTC and accelerated the biodegradation of CPAM. The diversity and composition of microbial community changed dramatically during HTC. Geobacillus, Thermobispora, Pseudomonas, Brevundimonas, and Bacillus were the dominant bacteria responsible for the high HTC efficiency. To our knowledge, this is the first study in which CPAM-containing sludge is treated using HTC. The ideal performance and the presence of key microorganisms revealed that HTC is feasible for the treatment of CPAM-containing sludge.

Identification of hyperthermophilic D-allulose 3-epimerase from Thermotoga sp. and its application as a high-performance biocatalyst for D-allulose synthesis.

D-Allulose 3-epimerase (DAE) is a vital biocatalyst for the industrial synthesis of D-allulose, an ultra-low calorie rare sugar. However, limited thermostability of DAEs hinders their use at high-temperature production. In this research, hyperthermophilic TI-DAE (Tm = 98.4 ± 0.7 ℃) from Thermotoga sp. was identified via in silico screening. A comparative study of the structure and function of site-directed saturation mutagenesis mutants pinpointed the residue I100 as pivotal in maintaining the high-temperature activity and thermostability of TI-DAE. Employing TI-DAE as a biocatalyst, D-allulose was produced from D-fructose with a conversion rate of 32.5%. Moreover, TI-DAE demonstrated excellent catalytic synergy with glucose isomerase CAGI, enabling the one-step conversion of D-glucose to D-allulose with a conversion rate of 21.6%. This study offers a promising resource for the enzyme engineering of DAEs and a high-performance biocatalyst for industrial D-allulose production.

Arsenic-contaminated soil remediation with hyperthermophilic compost: Effects on arsenic bioavailability, soil fertility and bacterial community.

Soil arsenic (As) contamination has posed a significant global environmental challenge seriously threatening human health. Compost has attracted broad interests as a kind of eco-friendly and versatile amendment. However, hyperthermophilic compost (HTC), which is newly-developed and more advantageous to environment, has not yet been widely utilized to remediate As-contaminated soil, and its effectiveness remains unclear. Herein, the effects of HTC amendment on soil fertility, As bioavailability, plant growth and soil bacterial community were investigated. After amended with HTC, soil nutrient content and enzyme activity were improved. Concurrently, the content of both total As and available As in soil was reduced, partially due to the formation of organo-As complex with the presence of humic acid and fulvic acid in HTC. Notably, Chinese white cabbage (Brassica campestris L. ssp. chinensis Makino) cultivated in HTC-treated soil exhibited better growth and less As uptake, but showed enhanced translocation of As from the below-ground part to the above-ground part. In particular, the lowest HTC addition ratio (HTC:Soil = 1:10, v:v) proved to be the most optimal, increasing the height, width and biomass of Chinese white cabbage from 9.92 ± 0.72 cm, 6.76 ± 0.31 cm and 4.43 ± 0.49 g, to 21.29 ± 0.48 cm, 19.3 ± 1.44 cm and 23.27 ± 2.45 g, respectively. The results of soil bacterial community analysis revealed that HTC amendment stimulated the growth and metabolism of soil microbes, augmenting the richness and diversity of bacteria related to the methylation and volatilization of As and plant growth. This work suggests that HTC can serve as an effective amendment for As-contaminated soil remediation, and a superior alternative to compound fertilizer for plant cultivation, displaying promising potential for agricultural applications.

Thermococcus thermotolerans sp. nov., a hyperthermophilic archaeon isolated from a chimney in the Southwest Indian Ocean.

An anaerobic hyperthermophilic archaeon was isolated from a black smoker chimney with a snail attachment at a water depth of 2 739 m in the Southwest Indian Ocean. The sample was taken from the chimney exterior wall. The enrichment was conducted under a continuous culture with temperature fluctuation of 80-130 °C over 24 h for 42 days at 28 MPa. The isolation was performed at 90 °C at 0.1 MPa. Cells of the isolated strain 813A4T were irregular cocci. Strain 813A4T grew at 60-94 °C (optimal growth at 85 °C) at 0.1 MPa, and growth was detected at up to 99 °C at 28 MPa. At 85 °C, the strain was able to grow at pressures ranging from 0.1 to 110 MPa (optimal pressure, 0.1-40 MPa). At 85 °C, the cells of 813A4T grew at pH 5.5-9 (optimal, pH 7.0) and a NaCl concentration of 1.0-4.0 % (w/v; optimum concentration, 2.5 % NaCl). Strain 813A4T utilized yeast extract, tryptone and peptone as single carbon sources for growth. Elemental sulphur stimulated its growth. The G+C content of the complete genome was 53.48 mol%. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain 813A4T belonged to the genus Thermococcus, with the highest sequence similarity to Thermococcus barossii SHCK-94T (99.73 %). The average nucleotide identity between strains 813A4T and SHCK-94T was 82.56 %. All these data indicated that strain 813A4T should be classified as representing a novel species of the genus Thermococcus, for which Thermococcus thermotolerans sp. nov. is proposed. The type strain is 813A4T (=JCM 39367T=MCCC M28628T).

Novelty three stages for humification of sewage sludge during hyperthermophilic aerobic fermentation.

Compared with conventional aerobic fermentation (CAF), there is limited knowledge of how hyperthermophilic aerobic fermentation (HAF) enhances the humification of sewage sludge. This study compared three novel stages of organic degradation, precursors, functional groups, bacterial community, and humus synthesis mechanism in HAF with CAF. The results showed that organic matter (OM) degraded rapidly, and 68% of the degradation could be completed of stage I in HAF. Compared with the initial stage, ammonium nitrogen (NH4+-N), water-soluble organic carbon, and water-soluble total nitrogen increased by 2.83 times, 40.5 times, and 33.5 times, respectively. Cellulose and hemicellulose decreased by 29.22% and 21.85%, respectively. These results suggested that temperature (>80 °C) and Bacillus dominated accelerate the humification process by rapidly improving OM degradation. Compared with the initial value of HAF, the maximum increment of reducing sugar at stage II was 297%, and the degradation rate of cellulose was effectively increased by 21.03% compared with that of CAF. The precursors such as reducing sugars and amino acids formed humus at stage II. The content of Aryl C increased significantly during the HAF process, the degree of polymerization of humus and the aromatization degree of HA and FA increased significantly, and complex organic macromolecular material polymers were formed at stage III. The sugar-amine condensation was the mechanism of humification in the sludge HAF process. This investigation provided three new stages of insights into the synthesis of humification during the HAF process and extended the current mechanism of humification in the HAF process.

Structures of filamentous viruses infecting hyperthermophilic archaea explain DNA stabilization in extreme environments.

Living organisms expend metabolic energy to repair and maintain their genomes, while viruses protect their genetic material by completely passive means. We have used cryo-electron microscopy (cryo-EM) to solve the atomic structures of two filamentous double-stranded DNA viruses that infect archaeal hosts living in nearly boiling acid: Saccharolobus solfataricus rod-shaped virus 1 (SSRV1), at 2.8-Å resolution, and Sulfolobus islandicus filamentous virus (SIFV), at 4.0-Å resolution. The SIFV nucleocapsid is formed by a heterodimer of two homologous proteins and is membrane enveloped, while SSRV1 has a nucleocapsid formed by a homodimer and is not enveloped. In both, the capsid proteins wrap around the DNA and maintain it in an A-form. We suggest that the A-form is due to both a nonspecific desolvation of the DNA by the protein, and a specific coordination of the DNA phosphate groups by positively charged residues. We extend these observations by comparisons with four other archaeal filamentous viruses whose structures we have previously determined, and show that all 10 capsid proteins (from four heterodimers and two homodimers) have obvious structural homology while sequence similarity can be nonexistent. This arises from most capsid residues not being under any strong selective pressure. The inability to detect homology at the sequence level arises from the sampling of viruses in this part of the biosphere being extremely sparse. Comparative structural and genomic analyses suggest that nonenveloped archaeal viruses have evolved from enveloped viruses by shedding the membrane, indicating that this trait may be relatively easily lost during virus evolution.

The Impact of Single-Stranded DNA-Binding Protein SSB and Putative SSB-Interacting Proteins on Genome Integrity in the Thermophilic Crenarchaeon Sulfolobus acidocaldarius.

The study of DNA repair in hyperthermophiles has the potential to elucidate the mechanisms of genome integrity maintenance systems under extreme conditions. Previous biochemical studies have suggested that the single-stranded DNA-binding protein (SSB) from the hyperthermophilic crenarchaeon Sulfolobus is involved in the maintenance of genome integrity, namely, in mutation avoidance, homologous recombination (HR), and the repair of helix-distorting DNA lesions. However, no genetic study has been reported that elucidates whether SSB actually maintains genome integrity in Sulfolobus in vivo. Here, we characterized mutant phenotypes of the ssb-deleted strain Δssb in the thermophilic crenarchaeon S. acidocaldarius. Notably, an increase (29-fold) in mutation rate and a defect in HR frequency was observed in Δssb, indicating that SSB was involved in mutation avoidance and HR in vivo. We characterized the sensitivities of Δssb, in parallel with putative SSB-interacting protein-encoding gene-deleted strains, to DNA-damaging agents. The results showed that not only Δssb but also Δalhr1 and ΔSaci_0790 were markedly sensitive to a wide variety of helix-distorting DNA-damaging agents, indicating that SSB, a novel helicase SacaLhr1, and a hypothetical protein Saci_0790, were involved in the repair of helix-distorting DNA lesions. This study expands our knowledge of the impact of SSB on genome integrity and identifies novel and key proteins for genome integrity in hyperthermophilic archaea in vivo.

Biochemical and mutational studies of an endonuclease V from the hyperthermophilic crenarchaeon Sulfolobus islandicus REY15A.

Endonuclease V (EndoV), which is widespread in bacteria, eukarya and Archaea, can cleave hypoxanthine (Hx)-containing DNA or RNA strand, and play an essential role in Hx repair. However, our understanding on archaeal EndoV's function remains incomplete. The model archaeon Sulfolobus islandicus REY15A encodes a putative EndoV protein (Sis-EndoV). Herein, we probed the biochemical characteristics of Sis-EndoV and dissected the roles of its seven conserved residues. Our biochemical data demonstrate that Sis-EndoV displays maximum cleavage efficiency at above 60 °C and at pH 7.0-9.0, and the enzyme activity is dependent on a divalent metal ion, among which Mg2+ is optimal. Importantly, we first measured the activation energy for cleaving Hx-containing ssDNA by Sis-EndoV to be 9.6 ± 0.8 kcal/mol by kinetic analyses, suggesting that chemical catalysis might be a rate-limiting step for catalysis. Mutational analyses show that residue D38 in Sis-EndoV is essential for catalysis, but has no role in DNA binding. Furthermore, we first revealed that residues Y41 and D189 in Sis-EndoV are involved in both DNA cleavage and DNA binding, but residues F77, H103, K156 and F161 are only responsible for DNA binding.

The Pyrococcus furiosus ironome is dominated by [Fe4S4]2+ clusters or thioferrate-like iron depending on the availability of elemental sulfur.

Pyrococcus furiosus is a hyperthermophilic anaerobic archaeon whose metabolism depends on whether elemental sulfur is (+S0) or is not (-S0) included in growth medium. Under +S0 conditions, expression of respiratory hydrogenase declines while respiratory membrane-bound sulfane reductase and the putative iron-storage protein IssA increase. Our objective was to investigate the iron content of WT and ΔIssA cells under these growth conditions using Mössbauer spectroscopy. WT-S0 cells contained ∼1 mM Fe, with ∼85% present as two spectroscopically distinct forms of S = 0 [Fe4S4]2+ clusters; the remainder was mainly high-spin FeII. WT+S0 cells contained 5 to 9 mM Fe, with 75 to 90% present as magnetically ordered thioferrate-like (TFL) iron nanoparticles. TFL iron was similar to chemically defined thioferrates; both consisted of FeIII ions coordinated by an S4 environment, and both exhibited strong coupling between particles causing high applied fields to have little spectral effect. At high temperatures with magnetic hyperfine interactions abolished, TFL iron exhibited two doublets overlapping those of [Fe4S4]2+ clusters in -S0 cells. This coincidence arose because of similar coordination environments of TFL iron and cluster iron. The TFL structure was more heterogeneous in the presence of IssA. Presented data suggest that IssA may coordinate insoluble iron sulfides as TFL iron, formed as a byproduct of anaerobic sulfur respiration under high iron conditions, which thereby reduces its toxicity to the cell. This was the first Mössbauer characterization of the ironome of an archaeon, and it illustrates differences relative to the iron content of better-studied bacteria such as Escherichia coli.

Adnaviria: a New Realm for Archaeal Filamentous Viruses with Linear A-Form Double-Stranded DNA Genomes.

The International Committee on Taxonomy of Viruses (ICTV) has recently adopted a comprehensive, hierarchical system of virus taxa. The highest ranks in this hierarchy are realms, each of which is considered monophyletic but apparently originated independently of other realms. Here, we announce the creation of a new realm, Adnaviria, which unifies archaeal filamentous viruses with linear A-form double-stranded DNA genomes and characteristic major capsid proteins unrelated to those encoded by other known viruses.

Self-assembly of Aeropyrum pernix bacilliform virus 1 (APBV1) major capsid protein and its application as building blocks for nanomaterials.

Virus capsid proteins have various applications in diverse fields such as biotechnology, electronics, and medicine. In this study, the major capsid protein of bacilliform clavavitus APBV1, which infects the hyperthermophilic archaeon Aeropyrum pernix, was successfully expressed in Escherichia coli. The gene product was expressed as a histidine-tagged protein in E. coli and purified to homogeneity using single-step nickel affinity chromatography. The purified recombinant protein self-assembled to form bacilliform virus-like particles at room temperature. The particles exhibited tolerance against high concentrations of organic solvents and protein denaturants. In addition, we succeeded in fabricating functional nanoparticles with amine functional groups on the surface of ORF6-81 nanoparticles. These robust protein nanoparticles can potentially be used as a scaffold in nanotechnological applications.

A thermophilic 8-oxoguanine DNA glycosylase from Thermococcus barophilus Ch5 is a new member of AGOG DNA glycosylase family

8-Oxoguanine (8oxoG) in DNA is a major oxidized base that poses a severe threat to genome stability. To counteract the mutagenic effect generated by 8oxoG in DNA, cells have evolved 8oxoG DNA glycosylase (OGG) that can excise this oxidized base from DNA. Currently, OGG enzymes have been divided into three families: OGG1, OGG2 and AGOG (archaeal 8oxoG DNA glycosylase). Due to the limited reports, our understanding on AGOG enzymes remains incomplete. Herein, we present evidence that an AGOG from the hyperthermophilic euryarchaeon Ch5 (Tb-AGOG) excises 8oxoG from DNA at high temperature. The enzyme displays maximum efficiency at 75°C-95°C and at pH 9.0. As expected, Tb-AGOG is a bifunctional glycosylase that harbors glycosylase activity and AP (apurinic/apyrimidinic) lyase activity. Importantly, we reveal for the first time that residue D41 in Tb-AGOG is essential for 8oxoG excision and intermediate formation, but not essential for DNA binding or AP cleavage. Furthermore, residue E79 in Tb-AGOG is essential for 8oxoG excision and intermediate formation, and is partially involved in DNA binding and AP cleavage, which has not been described among the reported AGOG members to date. Overall, our work provides new insights into catalytic mechanism of AGOG enzymes.

A packing for A-form DNA in an icosahedral virus.

Studies on viruses infecting archaea living in the most extreme environments continue to show a remarkable diversity of structures, suggesting that the sampling continues to be very sparse. We have used electron cryo-microscopy to study at 3.7-Å resolution the structure of the Sulfolobus polyhedral virus 1 (SPV1), which was originally isolated from a hot, acidic spring in Beppu, Japan. The 2 capsid proteins with variant single jelly-roll folds form pentamers and hexamers which assemble into a T = 43 icosahedral shell. In contrast to tailed icosahedral double-stranded DNA (dsDNA) viruses infecting bacteria and archaea, and herpesviruses infecting animals and humans, where naked DNA is packed under very high pressure due to the repulsion between adjacent layers of DNA, the circular dsDNA in SPV1 is fully covered with a viral protein forming a nucleoprotein filament with attractive interactions between layers. Most strikingly, we have been able to show that the DNA is in an A-form, as it is in the filamentous viruses infecting hyperthermophilic acidophiles. Previous studies have suggested that DNA is in the B-form in bacteriophages, and our study is a direct visualization of the structure of DNA in an icosahedral virus.

Genetic Study of Four Candidate Holliday Junction Processing Proteins in the Thermophilic Crenarchaeon Sulfolobus acidocaldarius.

Homologous recombination (HR) is thought to be important for the repair of stalled replication forks in hyperthermophilic archaea. Previous biochemical studies identified two branch migration helicases (Hjm and PINA) and two Holliday junction (HJ) resolvases (Hjc and Hje) as HJ-processing proteins; however, due to the lack of genetic evidence, it is still unclear whether these proteins are actually involved in HR in vivo and how their functional relation is associated with the process. To address the above questions, we constructed hjc-, hje-, hjm-, and pina single-knockout strains and double-knockout strains of the thermophilic crenarchaeon Sulfolobus acidocaldarius and characterized the mutant phenotypes. Notably, we succeeded in isolating the hjm- and/or pina-deleted strains, suggesting that the functions of Hjm and PINA are not essential for cellular growth in this archaeon, as they were previously thought to be essential. Growth retardation in Δpina was observed at low temperatures (cold sensitivity). When deletion of the HJ resolvase genes was combined, Δpina Δhjc and Δpina Δhje exhibited severe cold sensitivity. Δhjm exhibited severe sensitivity to interstrand crosslinkers, suggesting that Hjm is involved in repairing stalled replication forks, as previously demonstrated in euryarchaea. Our findings suggest that the function of PINA and HJ resolvases is functionally related at lower temperatures to support robust cellular growth, and Hjm is important for the repair of stalled replication forks in vivo.

A Novel Family of Winged-Helix Single-Stranded DNA-Binding Proteins from Archaea.

The winged helix superfamily comprises a large number of structurally related nucleic acid-binding proteins. While these proteins are often shown to bind dsDNA, few are known to bind ssDNA. Here, we report the identification and characterization of Sul7s, a novel winged-helix single-stranded DNA binding protein family highly conserved in Sulfolobaceae. Sul7s from Sulfolobus islandicus binds ssDNA with an affinity approximately 15-fold higher than that for dsDNA in vitro. It prefers binding oligo(dT)30 over oligo(dC)30 or a dG-rich 30-nt oligonucleotide, and barely binds oligo(dA)30. Further, binding by Sul7s inhibits DNA strand annealing, but shows little effect on the melting temperature of DNA duplexes. The solution structure of Sul7s determined by NMR shows a winged helix-turn-helix fold, consisting of three α-helices, three ß-strands, and two short wings. It interacts with ssDNA via a large positively charged binding surface, presumably resulting in ssDNA deformation. Our results shed significant light on not only non-OB fold single-stranded DNA binding proteins in Archaea, but also the divergence of the winged-helix proteins in both function and structure during evolution.

Enhanced Magnetic Hyperthermia of Magnetoferritin through Synthesis at Elevated Temperature.

Iron oxide nanoparticles have attracted a great deal of research interest in recent years for magnetic hyperthermia therapy owing to their biocompatibility and superior thermal conversion efficiency. Magnetoferritin is a type of biomimetic superparamagnetic iron oxide nanoparticle in a ferritin cage with good monodispersity, biocompatibility, and natural hydrophilicity. However, the magnetic hyperthermic efficiency of this kind of nanoparticle is limited by the small size of the mineral core as well as its low synthesis temperature. Here, we synthesized a novel magnetoferritin particle by using a recombinant ferritin from the hyperthermophilic archaeon Pyrococcus furiosus as a template with high iron atom loading of 9517 under a designated temperature of 90 °C. Compared with the magnetoferritins synthesized at 45 and 65 °C, the one synthesized at 90 °C displays a larger average magnetite and/or maghemite core size of 10.3 nm. This yields an increased saturation magnetization of up to 49.6 emu g-1 and an enhanced specific absorption rate (SAR) of 805.3 W g-1 in an alternating magnetic field of 485.7 kHz and 49 kA m-1. The maximum intrinsic loss power (ILP) value is 1.36 nHm2 kg-1. These results provide new insights into the biomimetic synthesis of magnetoferritins with enhanced hyperthermic efficiency and demonstrate the potential application of magnetoferritin in the magnetic hyperthermia of tumors.

Reduced pH is the primary factor promoting humic acid formation during hyperthermophilic pretreatment composting.

Hyperthermophilic pretreatment composting (HPC) has the advantages of enhanced composting efficiency and accelerated humic substance (HS) over conventional composting (CC). However, the mechanisms towards the accelerated humification process by HPC are still not clear. By means of sterilization technology, the roles of abiotic and biotic components on the formation of HS can be distinguished. The study investigated the humification degree and the succession of microbial community during HPC of pig manure. The mechanisms underlying the accelerated humification by HPC was identified using gamma sterilization. Results showed that HS content increased significantly by 13.72% in HPC and 29.93% in sterilized HPC inoculated with 1% CC (HPC_I), compared with 8.76% increase in CC and 7.12% increase in sterilized CC inoculated with 1% HPC during composting (CC_I). Compared with CC and CC_I, stronger intensities of HA-like and fulvic acid-like components were observed in HPC and HPC_I. Results showed that physicochemical properties, especially pH, were the key factors in accelerating the humification in HPC, while both physicochemical properties and microbial community contributed to the HA formation in CC. The study contributed to a better understanding of the mechanism towards the accelerated humification degree in HPC.

Isolation and Use of Coprothermobacter spp. to Improve Anaerobic Thermophilic Digestion of Grass.

The isolation of microorganisms was performed from digestate from the process of the anaerobic digestion (AD) of grass after hyperthermophilic pretreatment. The bacterium that was isolated and identified was Coprothermobacter sp. Using the isolated bacteria, an AD process on fresh grass (GB) and pretreated grass (PGB) was carried out with 10% of its addition. The highest methane yield of 219 NmlCH4/gVS was recorded for PGB at 55 °C. In contrast, fresh grass subjected to thermophilic digestion produced only 63 NmlCH4/gVS. Due to the addition of bacteria in the AD process, an increase in the efficiency of hydrogen and methane production was observed in both fresh grass and grass after pretreatment.