Efficient elimination of antibiotics and antibiotic resistance genes in hyperthermophilic sludge composting.

Composting is widely applied in recycling ever-increasing sewage sludge. However, the insufficient elimination of antibiotics and antibiotic resistance genes (ARGs) in conventional compost fertilizer poses considerable threat to agriculture safety and human health. Here we investigated the efficacy and potential mechanisms in the removal of antibiotics and ARGs from sludge in hyperthermophilic composting (HTC) plant. Our results demonstrated that the HTC product was of high maturity. HTC led to complete elimination of antibiotics and potential pathogens, as well as removal of 98.8 % of ARGs and 88.1 % of mobile genetic elements (MGEs). The enrichment of antibiotic-degrading candidates and related metabolic functions during HTC suggested that biodegradation played a crucial role in antibiotic removal. Redundancy analysis (RDA) and structural equation modelling (SEM) revealed that the reduction of ARGs was attributed to the decline of ARG-associated bacteria, mainly due to the high-temperature selection. These findings highlight the feasibility of HTC in sludge recycling and provide a deeper understanding of its mechanism in simultaneous removal of antibiotics and ARGs.

Efficient elimination of antibiotic resistome in livestock manure by semi-permeable membrane covered hyperthermophilic composting.

Livestock manure is a hotspot for antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs), and an important contributor to antibiotic resistance in non-clinical settings. This study investigated the effectiveness and potential mechanisms of a novel composting technology, semi-permeable membrane covered hyperthermophilic composting (smHTC), in removal of ARGs and MGEs in chicken manure. Results showed that smHTC was more efficient in removal of ARGs and MGEs (92% and 93%) compared to conventional thermophilic composting (cTC) (76% and 92%). The efficient removal in smHTC is attributed to direct or indirect negative effects caused by the high temperature, including reducing the involvement of bio-available heavy metals (HMs) in co-selection processes of antibiotic resistance, decreasing the bacterial abundance and diversity, suppressing the horizontal gene transfer and killing potential ARGs hosts. Overall, smHTC can efficiently remove the resistome in livestock manure, reducing the risk to crops and humans from ARGs residues in compost products.

Innovative hyper-thermophilic aerobic submerged membrane distillation bioreactor for wastewater reclamation.

For the first time, a hyper-thermophilic aerobic (>60 °C) bioreactor has been integrated with direct submerged membrane distillation (MD), highlighting its potential as an advanced wastewater treatment solution. The hyper-thermophilic aerobic bioreactor, operating up to 65 °C, is tailored for high organic removal, while MD efficiently produces clean water. Throughout the study, high removal rates of 99.5% for organic matter, 96.4% for ammonia, and 100% for phosphorus underscored the impressive adaptability of microorganisms to challenging hyper-thermophilic conditions and a successful combination with the MD process. Despite the extreme temperatures and substantial salinity accumulation reaching up to 12,532 µS/cm, the biomass of microorganisms increased by 1.6 times over a 92-day period, representing their remarkable resilience. The distillation flux ranged from 6.15 LMH to 8.25 LMH, benefiting from the temperature gradient in the hyper-thermophilic setting and the design of the tubular submerged MD membrane module. The system also excels in pH control, utilizing fewer alkali and nutritional resources than conventional systems. Meiothermus, Firmicutes, and Bacteroidetes, the three dominant species, played a crucial role, showcasing their significance in adapting to high salinity and decomposing organic matter.

Ignisphaera cupida sp. nov., a hyperthermophilic hydrolytic archaeon from a hot spring of Uzon (Kamchatka), and emended description of the genus Ignisphaera.

A novel strictly anaerobic hyperthermophilic archaeon, strain 4213-coT, was isolated from a terrestrial hot spring in the Uzon Caldera, Kamchatka (Russian Federation). Coccoid cells were present singly, in pairs, or aggregates, and occasionally were motile. The strain grew at 75-100 °C and within a pH range of 5.4-8.2 with the optimum at 92 °C and pH 6.4-6.7. Strain 4213-coT was a chemoorganoheterotroph, growing on proteinaceous substrates and mono-, di- and polysaccharides (starch, guar gum, xanthan gum). It did not require sodium chloride for growth. The complete genome of strain 4213-coT was 1.74 Mbp in size; its G+C content was 36.18 %. Genome analysis allowed to identify 25 genes encoding glycosidases involved in polysaccharide hydrolysis as well as genes of ADP-forming acetate-CoA ligase, lactate dehydrogenase and two [NiFe] hydrogenases responsible for acetate, lactate and hydrogen formation during fermentation. Moreover gene cluster encoding archaellum subunits was found. According to the phylogenomic analysis strain 4213-coT formed a species-level phylogenetic lineage within Ignisphaera genus. Our phylogenomic analysis also supports the delineation of the Ignisphaera genus into a separate family Ignisphaeraceae, as recently published. Here we propose a novel species Ignisphaera cupida, sp. nov. with type strain 4213-coT (=JCM 39446T=VKM B-3715T=UQM 41593T). Ecogenomic analysis showed that representatives of the Ignisphaera are thermophilic archaea, the majority of them were found in terrestrial hot springs and deep-sea hydrothermal vents. This study allowed a better understanding of physiology and ecology of Ignisphaeraceae - a rather understudied archaeal group.

Simultaneously improving the activity and thermostability of hyperthermophillic pullulanase by modifying the active-site tunnel and surface lysine.

Pullulanases are important starch-debranching enzymes that mainly hydrolyze the α-1,6-glycosidic linkages in pullulan, starch, and oligosaccharides. Nevertheless, their practical applications are constrained because of their poor activity and low thermostability. Moreover, the trade-off between activity and thermostability makes it challenging to simultaneously improve them. In this study, an engineered pullulanase was developed through reshaping the active-site tunnel and engineering the surface lysine residues using the pullulanase from Pyrococcus yayanosii CH1 (PulPY2). The specific activity of the engineered pullulanase was increased 3.1-fold, and thermostability was enhanced 1.8-fold. Moreover, the engineered pullulanase exhibited 11.4-fold improvement in catalytic efficiency (kcat/Km). Molecular dynamics simulations demonstrated an anti-correlated movement around the entrance of active-site tunnel and stronger interactions between the surface residues in the engineered pullulanase, which would be beneficial to the activity and thermostability improvement, respectively. The strategies used in this study and dynamic evidence for insight into enzyme performance improvement may provide guidance for the activity and thermostability engineering of other enzymes.

New insight into the spatio-temporal patterns of functional groups of hotspot inside the composting aggregates by synchrotron-based FTIR in hyperthermophilic composting.

Hyperthermophilic composting (HTC) is a recently developed and highly promising organic fraction of municipal solid waste (OFMSW) treatment technology. Investigation of organic matter (OM) dynamics in compost particle is thus crucial for the understanding of humification of HTC process. Herein, this work aimed to study the chemical and structural changes of OM at the molecular level during HTC of OFMSW using EEM and SR-FTIR analyses. Additionally, two-dimensional correlation spectroscopy (2D-COS) was also utilized to probe and identify the changes in chemical constituents and functional groups of organic compounds on the surface of compost particles during different composting periods. Results show that SR-FTIR can detect fine-scale (~µm) changes in functional groups from the edges to the interior of compost particles during different composting periods by mapping the particles in situ. In the hyperthermophilic stage (day 9), the extracted µ-FTIR spectrum reveals a distinct boundary between anaerobic and aerobic regions within the compost particle, with a thickness of anaerobic zone (1460 cm-1) of approximately 30 µm inside the particle's core. This provides direct evidence of anaerobic trends at compost microscales level within compost particles. 2D-COS analysis indicated that organic functional groups gradually agglomerated in the order of 1330 > 2930 > 3320 > 1600 > 1030 > 895 cm-1 to the core skeleton of cellulose degradation residues, forming compost aggregates with well physicochemical properties. Overall, the first combination of SR-FTIR and EEM provides complementary explanations for the humification mechanism of HTC, potentially introducing a novel methodology for investigating the environmental behaviors and fates of various organic contaminants associated with OM during the in-situ composting biochemical process.

The archaeal highly thermostable GH35 family ß-galactosidase DaßGal has a unique seven domain protein fold.

The most extensively studied ß-d-galactosidases (EC3.2.1.23) belonging to four glycoside hydrolase (GH) families 1, 2, 35, and 42 are widely distributed among Bacteria, Archaea and Eukaryotes. Here, we report a novel GH35 family ß-galactosidase from the hyperthermophilic Thermoprotei archaeon Desulfurococcus amylolyticus (DaßGal). Unlike fungal monomeric six-domain ß-galactosidases, the DaßGal enzyme is a dimer; it has an extra jelly roll domain D7 and three composite domains (D4, D5, and D6) that are formed by the distantly located polypeptide chain regions. The enzyme possesses a high specificity for ß-d-galactopyranosides, and its distinguishing feature is the ability to cleave pNP-ß-d-fucopyranoside. DaßGal efficiently catalyzes the hydrolysis of lactose at high temperatures, remains stable and active at 65 °Ð¡, and retains activity at 95 °Ð¡ with a half-life time value equal to 73 min. These properties make archaeal DaßGal a more attractive candidate for biotechnology than the widely used fungal ß-galactosidases.

A novel Mre11 protein from the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5 possesses 5'-3' exonuclease and endonuclease activities.

Mre11 is one of important proteins that are involved in DNA repair and recombination by processing DNA ends to produce 3'-single stranded DNA, thus providing a platform for other DNA repair and recombination proteins. In this work, we characterized the Mre11 protein from the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5 (Tba-Mre11) biochemically and dissected the roles of its four conserved residues, which is the first report on Mre11 proteins from Thermococcus. Tba-Mre11 possesses exonuclease activity for degrading ssDNA and dsDNA in the 5'-3' direction, which contrasts with other reported Mre11 homologs. Maximum degradation efficiency was observed with Mn2+ at 80 °C and at pH 7.5-9.5. In addition to possessing 5'-3' exonuclease activity, Tba-Mre11 has endonuclease activity that nicks plasmid DNA and circular ssDNA. Mutational data show that residues D10, D51 and N86 in Tba-Mre11 are essential for DNA degradation since almost no activity was observed for the D10A, D51A and N86A mutants. By comparison, residue D44 in Tba-Mre11 is not responsible for DNA degradation since the D44A mutant possessed the similar WT protein activity. Notably, the D44A mutant almost completely abolished the ability to bind DNA, suggesting that residue D44 is essential for binding DNA.

Effects of thermophilic bacteria inoculation on maturity, gaseous emission and bacterial community succession in hyperthermophilic composting.

Hyperthermophilic composting, characterized by temperatures equal to or exceeding 75 °C, offers superior compost maturity and performance. Inoculation with thermophilic bacteria presents a viable approach to achieving hyperthermophilic composting. This study investigates the effects of inoculating thermophilic bacteria, isolated at different temperatures (50 °C, 60 °C, and 70 °C) into compost on maturity, gaseous emissions, and microbial community dynamics during co-composting. Results indicate that the thermophilic bacteria inoculation treatments exhibited peak temperature on Day 3, with the maximum temperature of 75 °C reached two days earlier than the control treatment. Furthermore, these treatments demonstrated increased bacterial richness and diversity, along with elevated relative abundances of Firmicutes and Proteobacteria. They also fostered mutualistic correlations among microbial species, enhancing network connectivity and complexity, thereby facilitating lignocellulose degradation. Specifically, inoculation with thermophilic bacteria at 60 °C increased the relative abundance of Thermobifida and unclassified-f-Thermomonosporaceae (Actinobacteriota), whereas Bacillus, a thermophilic bacterium, was enriched in the 70 °C inoculation treatment. Consequently, the thermophilic bacteria at 60 °C and 70 °C enhanced maturity by 36 %-50 % and reduced NH3 emissions by 1.08 %-27.50 % through the proliferation of thermophilic heterotrophic ammonia-oxidizing bacteria (Corynebacterium). Moreover, all inoculation treatments decreased CH4 emissions by 6 %-27 % through the enrichment of methanotrophic bacteria (Methylococcaceae) and reduced H2S, Me2S, and Me2SS emissions by 1 %-25 %, 47 %-63 %, and 15 %-53 %, respectively. However, the inoculation treatments led to increased N2O emissions through enhanced denitrification, as evidenced by the enrichment of Truepera and Pusillimonas. Overall, thermophilic bacteria inoculation promoted bacteria associated with compost maturity while attenuating the relationship between core bacteria and gaseous emissions during composting.

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.

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.

Engineering hyperthermophilic pullulanase to efficiently utilize corn starch for production of maltooligosaccharides and glucose.

Pullulanase is a starch-debranching enzyme that hydrolyzes side chain of starch, oligosaccharides and pullulan. Nevertheless, the limited activities of pullulanases constrain their practical application. Herein, the hyperthermophilic type II pullulanase from Pyrococcus yayanosii CH1 (PulPY2) was evolved by synergistically engineering the substrate-binding pocket and active-site lids. The resulting mutant PulPY2-M2 exhibited 5-fold improvement in catalytic efficiency (kcat/Km) compared to that of PulPY2. PulPY2-M2 was utilized to develop a one-pot reaction system for efficient production of maltooligosaccharides. The maltooligosaccharides conversion rate of PulPY2-M2 reached 96.1%, which was increased by 5.4% compared to that of PulPY2. Furthermore, when employed for glucose production, the glucose productivity of PulPY2-M2 was 25.4% and 43.5% higher than that of PulPY2 and the traditional method, respectively. These significant improvements in maltooligosaccharides and glucose production and the efficient utilization of corn starch demonstrated the potential of the engineered PulPY2-M2 in starch sugar industry.

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.

Molecular Characterization of the Iron-Containing Alcohol Dehydrogenase from the Extremely Thermophilic Bacterium Pseudothermotoga hypogea.

Pseudothermotoga hypogea is an extremely thermophilic bacterium capable of growing at 90 °C and producing ethanol, which is catalyzed by an alcohol dehydrogenase (ADH). The gene encoding P. hypogea ADH (PhADH) was cloned, sequenced and over-expressed. The gene sequence (1164 bp) was obtained by sequencing all fragments of the gene, which were amplified from the genomic DNA. The deduced amino acid sequence showed high identity to iron-containing ADHs from other Thermotoga species and harbored typical iron- and NADP-binding motifs, Asp195His199His268His282 and Gly39Gly40Gly41Ser42, respectively. Structural modeling showed that the N-terminal domain of PhADH contains an α/ß-dinucleotide-binding motif and that its C-terminal domain is an α-helix-rich region containing the iron-binding motif. The recombinant PhADH was soluble, active, and thermostable, with a subunit size of 43 ± 1 kDa revealed by SDS-PAGE analyses. The recombinant PhADH (69 ± 2 U/mg) was shown to have similar properties to the native enzyme. The optimal pH values for alcohol oxidation and aldehyde reduction were 11.0 and 8.0, respectively. It was also thermostable, with a half-life of 5 h at 70 °C. The successful expression of the recombinant PhADH in E. coli significantly enhanced the yield of enzyme production and thus will facilitate further investigation of the catalytic mechanisms of iron-containing ADHs.

Enhanced lactic acid production from household food waste under hyperthermophilic conditions: Mechanisms and regulation.

An annual production of about 500 million tons of household food waste (HFW) has been documented, resulting in significant implications for human health and the environment in the absence of appropriate treatment. The anaerobic fermentation of HFW in an open system offers the potential to recover high value-added products, lactic acid (LA), thereby simultaneously addressing waste treatment and enhancing resource recovery efficiency. Most of LA fermentation studies have been conducted under mesophilic and thermophilic conditions, with limited research on the production of LA through anaerobic fermentation under hyperthermophilic conditions. This study aimed to produce LA through anaerobic fermentation from HFW under hyperthermophilic conditions (70 ± 1 °C), while varying pH values (5.0 ± 0.1, 7.0 ± 0.1, and 9.0 ± 0.1), and compare the results with LA production under mesophilic (35 ± 1 °C) and thermophilic (52 ± 1 °C) conditions. The findings of this study indicated that the combination of hyperthermophilic conditions and a neutral pH (pH7_70) yielded the highest concentration of LA, measuring at 17.75 ± 1.51 g/L. The mechanism underlying the high yield of LA at 70 °C was elucidated through the combined analysis of organics dissolution, enzymes activities, and 16S rRNA microbiome sequencing.

Biochemical characterization and mutational analysis of the NurA protein from the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5.

Archaeal NurA protein plays a key role in producing 3'-single stranded DNA used for homologous recombination repair, together with HerA, Mre11, and Rad50. Herein, we describe biochemical characteristics and roles of key amino acid residues of the NurA protein from the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5 (Tba-NurA). Tba-NurA possesses 5'-3' exonuclease activity for degrading DNA, displaying maximum efficiency at 45 °C-65 °C and at pH 8.0 in the presence of Mn2+. The thermostable Tba-NurA also possesses endonuclease activity capable of nicking plasmid DNA and circular ssDNA. Mutational data demonstrate that residue D49 of Tba-NurA is essential for exonuclease activity and is involved in binding ssDNA since the D49A mutant lacked exonuclease activity and reduced ssDNA binding. The R96A and R129A mutants had no detectable dsDNA binding, suggesting that residues R96 and R129 are important for binding dsDNA. The abolished degradation activity and reduced dsDNA binding of the D120A mutant suggest that residue D120 is essential for degradation activity and dsDNA binding. Additionally, residues Y392 and H400 are important for exonuclease activity since these mutations resulted in exonuclease activity loss. To our knowledge, it is the first report on biochemical characterization and mutational analysis of the NurA protein from Thermococcus.

Pleomorphic viruses establish stable relationship with marine hyperthermophilic archaea.

Non-lytic viruses with enveloped pleomorphic virions (family Pleolipoviridae) are ubiquitous in hypersaline environments across the globe and are associated with nearly all major lineages of halophilic archaea. However, their existence in other ecosystems remains largely unknown. Here, we show that evolutionarily-related viruses also infect hyperthermophilic archaea thriving in deep-sea hydrothermal vents. Archaeoglobus veneficus pleomorphic virus 1 (AvPV1), the first virus described for any member of the class Archaeoglobi, encodes a morphogenetic module typical of pleolipoviruses, including the characteristic VP4-like membrane fusion protein. We show that AvPV1 is a non-lytic virus chronically produced in liquid cultures without substantially affecting the growth dynamics of its host with a stable virus-to-host ratio of ~1. Mining of genomic and metagenomic databases revealed broad distribution of AvPV1-like viruses in geographically remote hydrothermal vents. Comparative genomics, coupled with phylogenetic analysis of VP4-like fusogens revealed deep divergence of pleomorphic viruses infecting halophilic, methanogenic, and hyperthermophilic archaea, signifying niche separation and coevolution of the corresponding virus-host pairs. Hence, we propose a new virus family, "Thalassapleoviridae," for classification of the marine hyperthermophilic virus AvPV1 and its relatives. Collectively, our results provide insights into the diversity and evolution of pleomorphic viruses beyond hypersaline environments.

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.

Molecular cloning and characterization of Pcal_0039, an ATP-/NAD+-independent DNA ligase from hyperthermophilic archaeon Pyrobaculum calidifontis.

The genome sequence of hyperthermophilic archaeon Pyrobaculum calidifontis contains an open reading frame, Pcal_0039, which encodes a putative DNA ligase. Structural analysis disclosed the presence of signature sequences of ATP-dependent DNA ligases. We have heterologously expressed Pcal_0039 gene in Escherichia coli. The recombinant protein, majorly produced in soluble form, was purified and functionally characterized. Recombinant Pcal_0039 displayed nick-joining activity between 40 and 85 °C. Optimal activity was observed at 70 °C and pH 5.5. Nick-joining activity was retained even after heating for 1 h at 90 °C, indicating highly thermostable nature of Pcal_0039. The nick-joining activity, displayed by Pcal_0039, was metal ion dependent and Mg2+ was the most preferred. NaCl and KCl inhibited the nick-joining activity at or above 200 mmol/L. The activity catalyzed by recombinant Pcal_0039 was independent of addition of ATP or NAD+ or any other nucleotide cofactor. A mismatch adjacent to the nick, either at 3'- or 5'-end, abolished the nick-joining activity. These characteristics make Pcal_0039 a potential candidate for applications in DNA diagnostics. To the best of our knowledge, Pcal_0039 is the only DNA ligase, characterized from genus Pyrobaculum, which exhibits optimum nick-joining activity at pH below 6.0 and independent of any nucleotide cofactor.