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Antioxidant-biocompatible and stable catalase-based gelatin–alginate hydrogel scaffold with thermal wound healing capability: immobilization and delivery approach

Hydrogel-based matrix prepared using biopolymers is a new frontier of emerging platforms for enzyme immobilization for biomedical applications. Catalase (CAT) delivery can be effective in inhibiting reactive oxygen species (ROS)-mediated prolongation of the wound healing process. In this study, to improve CAT stability for effective application, gelatin(Gel)–alginate (Alg) biocompatible hydrogel (Gel–Alg), as immobilization support, was prepared using calcium chloride as an ionic cross-linker. High entrapment efficiency of 92% was obtained with 2% Gel and 1.5% Alg. Hydrogel immobilized CAT (CAT–Gel–Alg) showed a wide range of pH from 4 to 9 and temperature stability between 20 to 60 °C, compared to free CAT. CAT–Gel–Alg kinetic parameters revealed an increased Km (24.15 mM) and a decreased Vmax (1.39 µmol H2O2/mg protein min) × 104. CAT–Gel–Alg retained 52% of its original activity after 20 consecutive catalytic runs and displayed improved thermal stability with a higher t1/2 value (half-life of 100.43 vs. 46 min). In addition, 85% of the initial activity was maintained after 8 weeks’ storage at 4 °C. At 24 h after thermal injury, a statistically significant difference in lesion sizes between the treated group and the control group was reported. Finally, our findings suggest that the superior CAT–Gel–Alg stability and reusability are resonant features for efficient biomedical applications, and ROS scavenging by CAT in the post-burn phase offers protection for local treatment of burned tissues with encouraging wound healing kinetics.

Introduction
Enzymes are outstanding biocatalysts that offer several remarkable advantages such as substrate specificity, operation under mild processing conditions, and specific biocompatible by-products (Abdel-Mageed et al. 2021a). Enzymes offer numerous promising applications in the biotechnological, biomedical, and pharmaceutical fields. Catalase (CAT) enzyme (EC 1.11.1.6) is an oxidoreductase enzyme that enables cells to remove reactive oxygen species (ROS) specifically hydrogen peroxide (H 2 O 2 ) that causes oxidative stress. CAT acts by breaking down hydrogen peroxide into water and molecular oxygen (Abdel-Mageed et al. 2012). When the concentration of ROS exceeds the cellular 1 3 73 Page 2 of 12 antioxidant defenses including ROS-scavenging enzymes, the oxidative stress is typically cytotoxic. Hence, there is a growing interest in the implications and the physiological significance of antioxidant CAT with a special interest in the development of biomedical and biotechnological products (Wang et al. 2021). CAT could enhance the therapeutic effects against ROS-induced acute kidney injury, acute liver injury, and wound healing through ROS removal (Wang et al. 2021). The pathological role of ROS in the inflammatory phase during wound healing is a result of the induced oxidative stress (Kurahashi and Fujii 2015). Previous studies have demonstrated the beneficial effect of CAT in wound healing (Abdel-Mageed et al. 2018a;Hu et al. 2017). However, efficient delivery of enzymes for therapeutic applications is usually hampered by its intrinsic low stability under operational conditions, high sensitivity to processing and storage conditions, and short half-life .
Immobilization is a well-established method for enzyme stabilization. Immobilization involves a chemical or a physical practice in which enzymes are fixed to or enclosed to support, generating a heterogeneous immobilized enzyme matrix that resembles the enzyme natural mode in biological cells. Selecting the best-suited immobilization support and the efficient methodology are the cornerstones for a successful immobilization process (Abdel-Mageed et al. 2019a). Physical entrapment is a favorable immobilization methodology that offers a simple procedure under mild conditions, low-cost, high activity recovery, and stability. Additionally, immobilization results in enhanced thermal and operational stability, possible enzyme recovery and reusability, and improved product yield, which are favorable for industrial applications (Abdel-Mageed et al. 2018b, 2019a. Hydrogels are a three-dimensional structured hydrophilic polymeric network made of hydrosols (water-absorbing natural such as alginate, and pectin or synthetic polymers such as polyethylene glycol and polyethylene oxide) that quickly swells in water retaining a significant amount (exceeds 90% water) within the network while preserving a semisolid consistency. In addition, hydrogels are presented as an advantageous matrix for CAT immobilization preserving its three-dimensional structure. Hydrogels including gelatin and alginate have been disclosed as ideal physicochemical mimetic biological scaffolds that can be used as a delivery vehicle for wound healing, as they can maintain a moist environment, absorb wound exudates, and fight bacteria (Sivaraj et al. 2021;Zhang et al. 2021).
Alginate is an anionic biopolymer obtained from brown algae that are usually used for hydrogel production and has been employed in several biomedical applications, protein/ drug delivery, tissue engineering, and wound healing (Aderibigbe and Buyana 2018; Miao et al. 2018). Sodium Alginate is composed of irregular blocks of β-d-mannuronic acid (M-block) and 1-4 linked α-l-guluronic residues (G-block) and offers distinct advantages over presently used wound dressing including improved absorption capacity of wound exudates, minimizes the risk of bacterial infections, reduces adverse allergic effects, and improves wound healing because of its biocompatibility, and non-immunogenicity. In addition, sodium alginate maintains humid surroundings that enhance reepithelization and fast granulation. It also displays hemostatic properties, which are beneficial for bleeding wounds (Zahid et al. 2021). For biomedical applications, sodium alginate offers a unique advantage where it forms a physically cross-linked hydrogel under mild conditions in the presence of divalent cations such as Ca 2+ , Fe 2+ or Mg 2+ , accompanied by a transition from solution to gel (Aderibigbe and Buyana 2018;Pilipenko et al. 2019). Among these cations, Ca 2+ is the most frequently used for ionic cross-linking of alginate, with calcium chloride (CaCl 2 ) demonstrated as one of the best Ca 2+ sources for this process. The cross-linking process is chiefly accomplished via the replacement of the sodium ions of G-blocks with the divalent cations such as Ca 2+ and bending of guluronic groups to create the egg box structure (Abasalizadeh et al. 2020). Alginates reported bio-adhesive properties and antibacterial activity is probably owed to the presence of free carboxyl groups (Cattelan et al. 2020).
Gelatin is a biocompatible and biodegradable polymer that is typically obtained from porcine, bovine, or fish collagen through acid or base hydrolysis (Liu et al. 2015). Gelatin has been recognized for wound healing application, as it possesses numerous substantial properties including plasticity, film forming and cell adhesion capacity, nonimmunogenicity, ability to synthesize in situ hydrogels, and tissue growth enhancement. A gelatin hydrogel is generally prepared through physical cross-linking in water at ≥ 2% w/v and ≤ 29 °C and usually dissolves at body temperature (37 °C), making it unsuitable for biomedical applications. Several cross-linking methods are used to enhance mechanical strength and provide the long-term stability of gelatin-based hydrogel. These include thermal gelation, ionic and covalent cross-linkers, Schiff base reactions, and enzyme-mediated cross-linking. Calcium ions (Ca 2+ ) play a significant role in improving the mechanical properties of hydrogels with ionically cross-linked networks (Abuelezz et al. 2020;Zhang et al. 2020). The ionic interaction between sodium alginate and gelatin occurs owing to the occurrence of ionizable amino and carboxyl groups, also due to the hydrogen interactions between the amine and carboxyl group (Li et al. 2021).
The post-burn inflammatory phase is accompanied by the massive production of ROS that is implicated in the progression of the local and distant inflammatory process (Vorauer-Uhl et al. 2002). Instantly following the thermal injury, polymorphonuclear leukocytes (PMNs) invade the lesion initiating the release of huge amounts of ROS in the interstitial fluid. Unfortunately, the concentration of antioxidant enzymes in the wound fluid is very low and insufficient to remove a large amount of ROS generated during the post-injury phase. In this regard, ROS acts directly on the cell membranes lipids (Saitoh et al. 1994). The beneficial effect of systemically administered antioxidant enzymes to prevent oxidative tissue injury is usually unsuccessful and is opposed by the bad pharmacokinetic profile of systemic enzyme administration (Steiling et al. 1999). In an attempt to find alternative strategies for successful biomedical use of CAT, a topical application using hydrogel is proposed as an effective means in inhibiting ROS-mediated impaired wound healing.
Accordingly, the integration of bioactive antioxidant CAT enzyme into Gel-Alg hydrogel emerges as a promising strategy to develop an antioxidant-efficient wound dressing. In this study, the first goal is to design biocompatible support for physical immobilization of CAT with high enzyme loading capacity, and high stability and reusability using a simple and mild methodology. The second goal is the use of the novel CAT-based hydrogel as a wound dressing matrix to prevent oxidative tissue damage for wound healing applications. Two naturally occurring hydrogels, gelatin (Gel) and alginate (Alg), were used as a support matrix for the physical immobilization of the CAT enzyme. Comparison of catalytic parameters such as temperature, pH, and kinetic parameters between the immobilized and the free enzyme was evaluated. Evaluation of the protective effect of CAT against ROS-induced wound damage was investigated using the thermal injury model.

Preparation of Gel-Alg hydrogels
The hydrogel matrix was prepared according to the previously published methodology (Rescignano et al. 2016). Briefly, sodium alginate and gelatin were firstly dissolved, separately, in potassium phosphate buffer, pH 7.0 at the concentration of 2% (w/v) gelatin and 1.5% (w/v) sodium alginate, followed by mixing the two obtained solutions under homogenization for 2 h at 1000 rpm with a mechanical stirrer. Then, the mixture solution was immersed into 3% CaCl 2 solution (w/v) at 30 °C for 30 min under low stirring and stored at 4 °C for 18 h to form the hydrogel matrix (Gel-Alg).

Determination of entrapment efficiency of CAT enzyme
Physical immobilization of CAT was achieved through the entrapment technique. In brief, 200 U mL −1 of CAT solution was prepared in 50 mM potassium phosphate buffer, pH 7.0, and was mixed in gelatin/alginate solution and the procedure was completed as described above to obtain CAT-based hydrogel (CAT-Gel-Alg).
Entrapment % was calculated according to the following Eq. (1):

Activity assay for free and immobilized CAT
CAT activity was assayed using a simple and quantitative spectrophotometric assay with H 2 O 2 as the substrate (Beers and Sizer 1952). The activity of CAT-Gel-Alg reaction was initiated by adding aliquots to a 3 mL of 20 mM H 2 O 2 solution prepared in 50 mM potassium phosphate buffer, pH 7.0, to obtain a final CAT concentration of 0.2-0.4 µg protein mL −1 in the assay reaction mixture. Then, the time course decomposition of the H 2 O 2 solution into molecular oxygen and water catalyzed by CAT enzyme at 25 °C was followed at 240 nm using a spectrophotometer (JASCO V-530, Japan). The calculations were performed based on the H 2 O 2 absorbance at 240 nm with the molar extinction coefficient of 43.6 M −1 cm −1 . For the free CAT activity measurements, the appropriate amount of the freshly diluted CAT samples was used as described above with a CAT concentration of 0.2-0.4 µg protein mL −1 in the assay reaction mixture. One unit of catalase is defined as the amount of enzyme that decomposes 1.0 µmol of H 2 O 2 into molecular oxygen and water per min under standard assay conditions. The protein concentration was determined using the Bradford method, with bovine serum albumin as a standard (Bradford 1976). (1) Total activity of immobilized CAT Total initial free enzyme activity × 100.

Water content
The hydrated sample was dried at 105 °C until constant weight and the sample water content was calculated according to the AOAC (2000) methods using Eq. (2): where W o and W 1 are the weight (g) of hydrogel before and after drying at 105 °C till constant weight, respectively.

Swelling behavior
The swelling behavior of the prepared hydrogel was determined by a gravimetric method as described by Zahid et al. (2021). The hydrogel samples were weighed and immersed in 100 mL of 0.1 M potassium phosphate buffer, pH 7.0, to determine the swelling behavior at 25 °C. At predetermined time intervals, the hydrogel was removed from the buffer solution by vacuum filtration and reweighed after wiping out the excess solution on the surface gently using a filter paper. The swelling ratio of the gel was calculated using Eq. (3): where W s and W i are the weight (g) of the hydrated swollen sample at time t (after intervals of 1, 2, 3, 4, 6, 8, and 12 h) and the weight (g) of the initial sample, respectively.

Thermal stability and half-life
The thermal stability of free and immobilized CAT was determined by measuring the residual catalytic activity at two temperatures (30 and 50 °C) in potassium phosphate buffer (0.1 M, pH 7.0) for different time intervals (15, 30, 45, 60 min). Afterward, enzyme activity was measured under standard assay conditions. The % residual CAT activity was calculated as the proportion of the residual activity retained about the initial activity at 25 °C, which was considered as 100%. The first-order inactivation rate constants K d were calculated according to the following Eq. (4): where A o is the initial enzyme activity and A is the residual activity after time t by plotting a graph of − ln A/A 0 on the Y-axis against time (t) on the X-axis. The slope of the graph gives K d of the enzyme and t 1/2 is calculated using Eq. (5):

pH and temperature
The pH profiles for free CAT and CAT-Gel-Alg were carried out at different pH values under standard assay conditions using the following buffers: 50 mM sodium acetate buffer for pH values 4.0 and 5.0 and 50 mM potassium phosphate buffer for pH values from 6.0 to 9.0 at 25 °C. The catalytic reactions were initiated by the addition of the appropriate dilution of free CAT or immobilized samples to the substrate solution. Optimum pH was considered to be at 100% catalytic activity and other activities were expressed as a percentage of this optimum 100% activity. The effect of temperature on free CAT and CAT-Gel-AGL was tested by assaying the catalytic activity after 15 min incubation at temperatures ranging from 20 to 60 °C with an interval of 10 °C. The residual enzyme activity was determined under standard assay conditions.

Kinetic parameters
Activity assays using H 2 O 2 were carried out for the determination of the intrinsic kinetic parameters (Michaelis-Menten constant (km)) and maximum reaction velocity (V max )) for the free CAT or CAT-Gel-AGL. Measurements were performed using different concentrations of H 2 O 2 ranging from 4 to 40 mM. The decomposition of each concentration of H 2 O 2 was initiated at a fixed overall CAT concentration of 0.2 µg mL −1 . The CAT activity assay was carried out under standard assay conditions.

Reusability study
The prepared CAT-Gel-Alg was employed to catalyze hydrogen peroxide decomposition reaction for several consecutive runs and CAT activity assay was carried out under standard conditions. After each reaction, the hydrogel was removed from the reaction mixture by filtration technique. Then, it was washed with potassium phosphate buffer (0.1 M, pH 7) at 25 °C three times to remove any residual substrate within the hydrogel matrix (Abdel-Mageed et al. 2012). The reusability study was performed for 20 consecutive runs and the obtained relative activities were plotted against the number of cycles.

Storage stability
Storage stability for both free CAT and CAT-Gel-Alg were determined at different time intervals after storage for 60 days in potassium phosphate buffer (0.1 M, pH 7.0) at 4 °C.

Experimental setup for thermal injury model
Adult male Wistar rats (250 ± 50 g) were divided into three groups (n = 8, total 24 animals), kept for 7 days of acclimatization and given a standard diet and water ad libitum. The experimental design was performed according to the method described by Vorauer-Uhl et al. (2001). Thermal burn was induced by exposing the depilated back skin of anesthetized animals [were anesthetized with ketamine (40 mg kg −1 ) and xylazine (20 mg Kg −1 )], placed in a supine position inside a plastic box open. The marked skin area was exposed to hot water (rats: 95 ± 1 °C, 15 s, 15 cm 2 ) causing a uniform second-degree burn. The experimental protocol was conducted in compliance with the Institutional Animal Care and Use Committee Guide 8th Edition 2011 (IACUC) National Research Centre, Cairo, Egypt, in accordance with the national and international Guide for the Care and Use of Laboratory Animals. Treatment with CAT-Gel-Alg hydrogel was performed immediately after thermal injury and twice daily for 3 days (treated group). In addition, one group was left untreated (naive group) and one was spread with the plain Gel-Alg hydrogel (control group). The size of the lesions and wound contracture were evaluated at 24, 48, and 72 h post-trauma. The size of the lesions was determined using a planimetric method that projects the lesions' shapes onto a transparent pattern foil.

Statistical analysis
All experiments were carried out in triplicate. The data were analyzed using one-way analysis of variance followed by the Student's t test for comparisons between groups (SPSS 19.0 was used to perform statistical analysis). Significance was indicated by p < 0.05 in this study.

Preparation of Gel-Alg hydrogel
Using a simple and mild methodology Gel-Alg was successfully prepared at 2% W/V gelatin and 1.5% sodium alginate. Alginate hydrogels are typically formed after cross-linking in an aqueous medium in the presence of divalent cations such as Ca 2+ that acts as ionic cross-linking agents. The high aqueous solubility of CaCl 2 made it one of the most commonly employed cross-linking agents in aqueous media. It is suggested that the Ca 2+ interacts with the G-block of the biopolymer by replacing the sodium ions of the G-blocks forming the known egg box structure. On the other hand, the M-blocks of alginate exhibit weak interactions with calcium cations (Rescignano et al. 2016). The concentration of the gelling agent, mixing time, speed and temperature were optimized in preliminary studies (data not shown). These factors should be carefully chosen as they affect gel uniformity and strength.
Uniform hydrogels with improved mechanical strength are usually produced by decreasing the rate of gelation. A potassium phosphate buffer was used to slow and control the gelation rate, as the phosphate groups in the buffer compete with the -COOH group of alginate during the reaction with calcium ions. In addition, decreasing the temperature lowers the divalent cations' reactivity, which decreases the gelation rate resulting in a highly ordered cross-linked network that consequently improves the mechanical properties of the prepared hydrogel (Abasalizadeh et al. 2020). To overcome the alginate in situ limitations, gelatin was used in conjugation to prevent a reduction in viscosity and dissolution upon application to biological tissues. In addition, calcium chloride was used as the sole crossing agent and no organic solvents were employed during the preparation of the hydrogel to preserve the fragile nature of CAT.

Immobilization of CAT
CAT is a highly valuable antioxidant enzyme that has great potentials for biomedical applications. Physical immobilization technique through entrapment is a mild process that does not affect the enzyme structure and hence its catalytic activity. The entrapment efficiency of CAT in Gel-Alg hydrogel was found to be 92% (Table 1). It is expected that under the mild conditions employed in hydrogel preparation and the absence of organic solvents, the CAT structure in biopolymer gel is unchanged. Additionally, it has been reported before that chemical cross-linkers such as glutaraldehyde adversely affect enzyme activity (Gür et al. 2017). High entrapment efficiency upon physical immobilization has been reported by other authors using different carriers such as hydrogels (85%, Jiang and Zhang1993), chitosan beads (96%, Kaushal et al. 2018), and vesicles (≥ 80%, Abdel-Mageed et al. 2012). Additionally, it is worth noting that both sodium alginate and gelatin act directly as CAT stabilizers; CAT, alternatively, prevents degradation of the hydrogel either by light or by oxidation, avoiding possible chemical reactions.

Water content of the hydrogels
The hydrogel water content is a significant measure to determine water absorbing and retaining capacity. The prepared hydrogels (Gel-Alg and CAT-Gel-Alg) exhibited a high capacity to absorb and retain water with no significant difference in behavior. The water content of Gel-Alg and CAT-Gel-Alg was 95.72 and 96.1%, respectively. These results are an indication of the ability of the hydrogel to prevent the accumulation of wound fluid by absorbing the exudates for effective wound dressing (Stephen 2004;Cattelan et al. 2020).

Swelling behavior
Hydrogel swelling behavior is a primary criterion for investigating the ability to absorb water and can be utilized to verify its efficiency to protect enzyme conformational structure upon immobilization. The swelling behavior of Gel-Alg and CAT-Gel-Alg sample hydrogels as a function of time is shown in Fig. 1. Both Gel-Alg and CAT-Gel-Alg samples revealed a timedependent swelling profile, where they started to absorb water soon after they were dropped into the deionized water with faster absorption in the first 2 h of immersion and attained a hydration equilibrium point after 3 h ( Fig. 1). Gel-Alg and CAT-Gel-Alg reached a maximum absorption value of 660% and 674%, respectively, after 3 h. Similarly, Muanruksa et al. (2020) reported that the prepared alginate hydrogel absorbed a greater amount of water, as the retention time increased till an equilibrium status was reached (3 h). These results are an indication of the ability of the hydrogel to prevent the accumulation of wound fluid by absorbing the exudates for effective wound dressing.

Effect of temperature and thermal stability on CAT activity
Determination of thermal stability is highly essential for effective enzyme application. Figure 2 represents the thermal stability of both free and encapsulated CAT as a function of temperature. CAT activity was assayed after enzyme incubation at different temperatures ranging from 20 to 60 °C for 15 min preceding the substrate addition at pH 7.0. It is being found that both free and immobilized CAT exhibited the highest activity at 40 °C. In addition, they both showed more stability at a lower temperature between 20 and 40 °C. CAT-Gel-Alg exhibited significant stability at higher temperatures where 55% of activity was retained at 60 °C. The activity of the free CAT started to steeply decline after 40 °C and reached 28% at 60 °C. It is typically reported that immobilized enzymes exhibit improved thermal stability than the free enzyme due to the reduction of conformational flexibility in the immobilized enzyme. Similarly, Kaushal et al. (2018) and Abdel-Mageed et al. (2012) reported the improved thermal stability of CAT upon immobilization. Table 2 shows the values of inactivation rate constants K d as evaluated from the slope of the plot and t 1/2 for free and immobilized CAT. At 50 °C the calculated K d for free CAT and CAT-Gel-Alg was 0.015and 0.0069 min −1 , respectively. The results show that the immobilization procedure adopted in this study improved the thermal stability of CAT as evident from t 1/2 values. The t 1/2 values at 50 °C demonstrate the preserving effect of immobilization on CAT activity with t 1/2 values of 46 and 100 min for free CAT and CAT-Gel-Alg, respectively. Thermal inactivation and degradation of enzymes regularly take place at higher temperatures thus exhibiting lower t 1/2 values (Abdel-Mageed et al. 2019b). Extensive application opportunities are expected for immobilized enzymes with improved thermal stability. Several authors have reported the improved thermal stability of CAT upon immobilization where the polymeric network serves to preserve the conformational structure of the enzyme against denaturation (Gur et al. 2017;Kaushal et al. 2018). These results also suggest that the chosen biopolymers in this study did not exert any denaturation effect on CAT even under thermal stress. This is probably because of the physical entrapment of CAT within the polymer network, the effect of the cross-linking process, and the possible protective effect of calcium in the thermostability of CAT (Arica et al. 1999).

Effect of pH on the CAT catalytic activity
The effect of pH on free CAT and immobilized CAT was studied to serve as an indication of the change in the chemical characterization of CAT upon immobilization. The effect of pH on enzyme activity for free CAT and CAT-Gel-Alg forms was studied with a 4-9 pH range using different buffers. Both free and immobilized CAT exhibited maximum catalytic activity at pH 7.0 using potassium phosphate buffer. At all measured pH values, CAT-Gel-Alg exhibited higher activity in comparison to the free form (Fig. 3). These improved stability results can be credited to the physicochemical stabilization of the enzyme inside the hydrogel matrix. Enzyme immobilization typically impacts the enzyme ionization state, its dissociation, and its conformational structure. Hence, an influential effect of pH is usually observed that indicates an association between the pH, stability, and the catalytic activity of the immobilized enzymes (Abdel-Mageed et al. 2019a).

Kinetic parameters
The velocity of an enzyme reaction is ultimately affected by the substrate concentration of that specific enzyme. This relationship is described by Michaelis-Menten kinetics, given by the following equation: where K m is the Michaelis-Menten constant and V max is the maximum reaction rate. The K m is calculated using the Lineweaver-Burk plot which presents a linear relationship between 1/V and 1/S, respectively. The kinetic parameters in this study were determined by varying the H 2 O 2 substrate concentration for both free and immobilized CAT. The calculated parameters for free and CAT-Gel-Alg are presented in Table 3. The K m value (24.15 mM) for CAT-Gel-Alg was higher than that of the free CAT (20.5 mM). The obtained K m for CAT-Gel-Alg suggests a decrease in the affinity of the enzyme toward substrate upon entrapment in the hydrogel matrix, i.e., hydrogen peroxide in comparison to free CAT. As presented in Table 3, the V max of immobilized CAT (13,900 μmol H 2 O 2 /mg protein min), was slightly lower than that of free CAT (14,700 μmol H 2 O 2 / mg protein min). The reason for the higher V max of the free CAT than the immobilized one can be explained based on the existence of diffusional constraints that restrict substrate access to the enzyme upon immobilization into the hydrogel matrix (Abdel-Mageed et al. 2021b). In general, the catalytic efficiency of CAT is preserved at a higher level after immobilization in comparison to the free CAT. Similarly, Başak and Aydemir (2013)

Reusability and operational stability
The reuse of immobilized enzymes is an important attribute that is highly significant for industrial applications. Results revealed that CAT-Gel-Alg exhibits superior reusability features, as it retained almost all the original activity for seven consecutive cycles. Afterward, a gradual decrease in activity occurred with 52% residual activity after the 20 consecutive runs of catalytic reactions. In contrast, the relative  activity of free CAT gradually decreased until it reached 42% relative activity at run 13 (Fig. 4). The loss of enzymatic activity after repeated use is related to possible conformational changes as a result of consecutive use of the enzyme and the possible poisoning effect of the substrate (Arica et al. 1999). The enzyme was also leached from the immobilization matrix during the washing process. These effects result in alterations in the tertiary structure of the enzyme, which is the primary reason for enzyme deactivation (Abdel-Mageed et al. 2012). It can be concluded that the immobilization procedure used in this study achieved the goal of preserving high enzyme catalytic efficiency for biomedical applications.

Storage stability
Storage stability is a key factor for the practical application of immobilized enzymes. Free CAT and immobilized Gel-Alg-CAT were stored at 4 °C in potassium phosphate buffer (0.1 mM, pH 7.0) and enzyme activity was carried out at predetermined time intervals for 60 days. Typically, enzymes exhibit short-term stability in the free form. Figure 5 demonstrates that CAT-Gel-Alg activity gradually decreased in comparison to the free form. After 60 days, CAT activity was assayed and 26 and 85% of residual enzyme activity were detected for the free enzyme and immobilized CAT, respectively. These results highlight the superior stability and advantages of immobilized CAT onto hydrogel. The superior stability of CAT upon immobilization has been reported by Kaushal et al. (2018) with 70% residual activity after 60 days' storage.

CAT-Gel-Alg efficiency in wound healing
Immediately following burn injury, the inflammatory processes propagated adversely affect the wound healing process. However, following thermal injury, production of short-lived highly reactive oxygen species upsurges in the interstitial fluid with the increase in tissue ischemia, which results in extended cellular damage. Hence, the pathophysiological events following the radical-mediated injury can be avoided via free radical scavengers or inhibitors (Vorauer-Uhl et al. 2002). Given these findings, we investigated the usefulness of CAT administered topically and immediately after thermal burning with hot water. The study was focused on the visible changes implicated by CAT-Gel-Alg administered topically via spreading onto the aggrieved tissues. A scalding burn was elected as a simple procedure to produce uniform consistent burns through hot water exposure. Following the thermal burn induction, all animal groups showed a statistically identical degree of inflammation. In Table 4 the average lesion size areas of each group are presented. 24 h post-trauma, the lesion sizes of the untreated animals (group A) expanded to 155% and of those treated with plain gel (B) to 128% in respect of the initial area (100%). Lesion sizes of the CAT-Gel-Alg treated groups expanded less, 102% in group C. During the first 24 h, group (C) treated with CAT-Gel-Alg exhibited a statistically significant (p ≤ 0.05) lower dilatation of lesions in comparison to the untreated animals (A). Additionally, a significant difference (p ≤ 0.05) between the group treated with the plain gel (B) and with CAT-Gel-Alg (C) was After an initial lag time of 1 day, the rate of wound healing was monitored visually by subjective evaluation using two independent inspectors. The inspectors were not informed about the treated group to avoid bias in the assessment. Significant differences in wound healing were observed visually after 48 h for the CAT-Gel-Alg treated groups. Additionally, the treated group showed a reduction in lesion size and an enhanced rate of wound healing after 72 h. It was previously reported that the high water content of hydrogels is perfect for wound hydration and supports conditions in the wound bed for accelerated angiogenesis and even relieves pain (Field and Kerstein 1994). In conclusion, our data suggest that CAT-Gel-Alg is a potent radical scavenger that exhibited a protective effect upon administration in a suitable formulation that is applied immediately after trauma. Whether the use of higher enzyme concentration results in superior edema attenuation needs further investigations.

Conclusion and future prospects
This research aimed to synthesize a safe, sustainable, and biocompatible hydrogel of gelatin and sodium alginate with co-incorporation of catalase enzyme for application in wound healing. A simple methodology was employed in this study and was carried out under mild conditions with no high energy input and excluding the use of any organic solvents. In this work, the immobilized CAT exhibited high thermal and storage stability and retained approximately 52% of its relative activity after the 20th catalytic run. Natural gelatin and sodium alginate biopolymers are cost-efficient and proved to offer effective physical immobilization

Series1 Series2
Free CAT CAT-Gel-Alg Table 4 The effect of CAT-Gel-Alg treatment on the change in lesion size following burn wound induction The mean size of lesions was calculated and results are expressed as average means and standard deviation in cm 2 at 24 h. Values are means ± SD a The percentage increases were calculated in relation to the initial scalded lesion size of 20 cm 2 b Means are significantly different from native and control at (p ≤ 0.05)) of CAT. Finally, the data obtained in this study suggest a promising effect of topical treatment of thermal injury using antioxidant CAT-Gel-Alg that would offer protection for burned tissues with a promising wound healing process. For future application though, additional investigations are needed to determine the optimal CAT-based hydrogel matrix dosage. It is expected that the outcome of the ongoing and recent advancements in this field will yield a superior and efficient generation of biomaterials with novel possibilities for different biomedical uses.

Conflict of interest
The authors declare that they have no conflict of interest in the publication.
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An essential role for tungsten in the ecology and evolution of a previously uncultivated lineage of anaerobic, thermophilic Archaea

Trace metals have been an important ingredient for life throughout Earth’s history. Here, we describe the genome-guided cultivation of a member of the elusive archaeal lineage Caldarchaeales (syn. Aigarchaeota), Wolframiiraptor gerlachensis, and its growth dependence on tungsten. A metagenome-assembled genome (MAG) of W. gerlachensis encodes putative tungsten membrane transport systems, as well as pathways for anaerobic oxidation of sugars probably mediated by tungsten-dependent ferredoxin oxidoreductases that are expressed during growth. Catalyzed reporter deposition-fluorescence in-situ hybridization (CARD-FISH) and nanoscale secondary ion mass spectrometry (nanoSIMS) show that W. gerlachensis preferentially assimilates xylose. Phylogenetic analyses of 78 high-quality Wolframiiraptoraceae MAGs from terrestrial and marine hydrothermal systems suggest that tungsten-associated enzymes were present in the last common ancestor of extant Wolframiiraptoraceae. Our observations imply a crucial role for tungsten-dependent metabolism in the origin and evolution of this lineage, and hint at a relic metabolic dependence on this trace metal in early anaerobic thermophiles.

E vidence is mounting that some of the most ancient life forms inhabited terrestrial hot springs present on the anoxic early Earth [1][2][3][4] . Such habitats could have mobilized and concentrated nutrients including trace metals essential to thermophiles, but that differ from the metals essential to extant aerobic mesophiles 4 . Contemporary terrestrial hot springs are reasonable analogs for those on early Earth, often exhibiting low reduction potentials and sulfur-rich anoxic conditions 5 . In the presence of sulfide, several trace metals, including molybdenum (Mo), copper (Cu), nickel (Ni), and cobalt (Co) react with sulfide and precipitate as solid phases 6 , rendering them inaccessible to microbial cells. This, in turn, could have represented a strong selective pressure to evolve metalloenzymes that were dependent on less thiophilic trace metals, such as manganese (Mn) and tungsten (W).
In modern hot springs, most microbial lineages lack a cultured representative 7,8 , making the assessment of nutrient requirements difficult. Although cultivation-independent "omics" techniques hold tremendous promise in assessing the functional potential and activity of communities in their natural state, results from such studies are limited by what is known of characterized proteins from cultured organisms. Yet, for uncultivated lineages, many of the proteins are poorly annotated, often leading to an incomplete picture of their ecology and physiology. As such, there is a great need to bring these organisms into the culture so that the traits that permit the habitation of terrestrial hot springs, both today and in the geological past, can be examined.
Candidatus Caldarchaeales (syn. Aigarchaeota) is a yetuncultivated lineage detected in high-temperature environments that was first described based on a genomic assembly from a thermal stream in a subsurface gold mine in Japan 9,10 . Although originally designated as candidate phylum Aigarchaeota, this lineage has been reassigned to the order Ca. Caldarchaeales within the phylum Thermoproteota and class Nitrososphaeria 11 . For simplicity, we use this latter taxonomy without the Candidatus designation for uncultivated taxa. Caldarchaeales are commonly found in hydrothermal and geothermal ecosystems 8 , where they can be abundant [12][13][14] . Metagenomics and 16S rRNA gene amplicon surveys have previously shown a high abundance of Caldarchaeales in sediments of Great Boiling Spring (GBS, Nevada, USA) 15 . In particular, members of the putative genus "Aigarchaeota Group 4" (AigG4) 8 were abundant in in-situ GBS enrichments cultivated on lignocellulosic substrates such as corn stover 16,17 . AigG4 species from GBS and from hot springs in Tengchong, China (GMQ_bin_10 and JZ_bin_10 in ref. 12 ) have been predicted to be anaerobic heterotrophs that degrade polysaccharides and peptides 13,14 . However, these predictions have yet to be experimentally validated and other physiological traits of AigG4 remain unknown.
Here, we report the enrichment and stable laboratory growth of an AigG4 species from GBS we designate Wolframiiraptor gerlachensis. After initial unsuccessful attempts to grow the species without amending growth media with spring water, analysis of a metagenome-assembled genome (MAG) of W. gerlachensis suggested possible dependence on the biologically rare trace metal W, as evidenced by six annotated W-dependent ferredoxin oxidoreductases that could play central roles in anaerobic carbohydrate degradation. Indeed, stable cultivation of W. gerlachensis in a synthetic medium containing corn stover or a monosaccharide mix required the addition of W. NanoSIMS analysis demonstrated that xylose is the carbohydrate monomer preferred by W. gerlachensis in in-situ corn stover-degrading enrichments. Based on these experimental results and the genomic potential of the MAG, we propose that W is essential for carbohydrate metabolism and plays a critical role in one or more of the annotated W-dependent ferredoxin oxidoreductases, which could replenish reducing equivalents for a type-4 [NiFe] membrane-bound hydrogenase, producing a proton motive force. Using environmental metagenomes from three continents and a marine hydrothermal vent, we present evidence that both putative tungstate (WO 4 2-) transporters and tungstoenzymes were ancestral in this lineage, herein designated the family Wolframiiraptoraceae, and trace their evolution among 78 highquality MAGs representing four genera and 11 species. While it is known that W is important for several archaeal groups [18][19][20][21] , including methanogens, representatives of the Thermococcales and Thermoproteales are the only known archaea that require W for growth 18 . Our investigation is unique in showing long-term W-dependent growth dynamics in a mixed culture, identifying W-requiring archaea outside of the Methanobacteriota (formerly Euryarchaeota), and demonstrating large-scale expansions of W-dependent enzymes in a microbial lineage.

Results
A stable enrichment culture of an anaerobic member of the Caldarchaeales. Because W. gerlachensis was previously enriched on corn stover incubated in GBS 17 , we deployed new corn stover in-situ enrichments and incubated them for 6 months (Supplementary Fig. 1). The water above the incubation site was 86.5°C, with a pH of 7.31 at the time the enrichment was deployed. After incubation, W. gerlachensis was present in the in-situ enrichment at~3% of the total community based on 16S rRNA gene tag analysis (Fig. 1a), similar to previous results 17 , along with other taxa previously observed in enrichments or GBS sediments 16,17 . Based on the reproducible enrichment of W. gerlachensis on corn stover, and the genome-predicted anaerobic lifestyle 13,14 , we used the in-situ enrichment to inoculate 13 different anaerobic media that contained corn stover and/or xyloglucan and keratin as carbon sources. Some enrichments also contained previously sampled GBS spring water that was filtered (0.2 µm) and autoclaved at a 1:1 ratio with synthetic medium (Supplementary Table 1) to test if unknown components of the spring water were required for growth. The abundance of W. gerlachensis under culture conditions without added spring water dropped below the level of detection. However, W. gerlachensis was maintained after multiple transfers in two cultures with added spring water (A8 and A10) at concentrations of~0.25-1.0 × 10 6 16S rRNA gene copies mL -1 culture, with a relative abundance of~0.5-7% (Fig. 1b).
The two culture conditions that maintained stable populations of W. gerlachensis contained GBS spring water but were otherwise identical to synthetic media enrichments where W. gerlachensis was not maintained (compare A10 with A1, and A8 with A3; Supplementary Table 1). Additional experiments determined that nitrate (0.1 mM) was inhibitory to W. gerlachensis (explaining the lack of growth in culture A9) and that robust growth was dependent on anoxic conditions and on the presence of corn stover rather than other substrates (Supplementary Figs. 1 and 2  and Supplementary Table 1). As a result, most subsequent experiments were performed using a variation of the A10 medium (A10X2) with corn stover as the main carbon source under anoxia. Under these conditions, W. gerlachensis has been maintained at an abundance of 10 6 -10 7 16S rRNA gene copies mL -1 culture for over 4 years. To our knowledge, these represent only the second reported laboratory cultures containing members of the Caldarchaeales 22 .
W is essential to maintain the growth of W. gerlachensis. Since GBS spring water was necessary for the maintenance of W. gerlachensis cultures ( Fig. 1b and Supplementary Table 1), we compared the composition of the synthetic medium to GBS spring water using inductively coupled plasma mass spectrometry (ICP-MS) (Supplementary Data 1). Biologically relevant trace elements in the GBS spring water that were absent in the synthetic medium trace metal solution (Supplementary Data 2) included Ni (2.2 nM), which was however in the complete corn stover medium (19-26 nM), and W (263 nM) 23 . Only W was present in the spring water and not elsewhere, such as in the substrate (~0.11 nM) or media components (<0.05 nM, Supplementary Data 2). Furthermore, preliminary analysis of a W. gerlachensis MAG obtained from the laboratory cultures indicated the presence of multiple W-associated enzymes and WO 4 2-ABC transporters. We therefore hypothesized that W was the key component in the GBS spring water allowing maintenance of W. gerlachensis.
The utilization of W was conceivable given its previous recognition as an essential element for diverse ferredoxin oxidoreductases in the marine heterotrophic hyperthermophile Pyrococcus furiosus (Thermococcales) [24][25][26][27][28] . Nevertheless, W biochemistry is rare in microorganisms, although the low redox potential of W cofactors makes them especially suitable for anaerobes 29 . For instance, the low redox potential of the W 6+ /W 4+ couple makes it preferable to Mo, which is more bioavailable in oxic environments and serves equivalent cellular functions in formaldehyde oxidoreductases and benzoyl CoA reductases 30,31 . In acetylene hydratase, W enables sharing of electrons of the outer orbital shell with the substrate and provides electrostatic stabilization to transition states 32 . In GBS, W is likely derived from minerals of the Scheelite (CaWO 4 ) and Wolframite groups ([Fe, Mn]WO 4 ), within W-rich deposits overlaying Triassic clays that can be extensive in the Great Basin 33 .
We confirmed that W. gerlachensis is dependent on W using a synthetic medium with discrete additions of WO 4 2- (Fig. 2). After two transfers in a synthetic medium without W amendment, W. gerlachensis fell to very low or undetectable levels based on amplicon sequence variant (ASV) counts, whereas addition of 0.1 or 2 µM WO 4 2alleviated this effect (Fig. 2a). Importantly, the addition of W to the synthetic medium led to W. gerlachensis abundances indistinguishable from those in the original spring water-containing medium (ANOVA, p > 0.05). These results were also reflected in W. gerlachensis absolute abundance as determined by qPCR. The omission of W induced a drop of W. gerlachensis 16S rRNA genes by 2-3 orders of magnitude (Fig. 2b). Tungsten starvation induced loss of W. gerlachensis earlier (after the second transfer) in corn stover relative to sugar mix cultures ( Fig. 2c and Supplementary Fig. 3), with orders-ofmagnitude differences in W. gerlachensis 16S rRNA genes with varying W concentrations. Concentrations of 20 nM WO 4 2yielded higher W. gerlachensis 16S rRNA gene numbers than 5 nM WO 4 2-(corn stover) and 1 nM WO 4 2-(sugar mix, ANOVA, p < 0.05 for both), suggesting 20 nM WO 4 2was the critical threshold to maintain W. gerlachensis over at least three transfers in synthetic medium (Fig. 2c). We should note that, given~0.8 mM sulfide added to the medium as reducing agent, the dominant W species is likely WS 4 2-, which microbial cells would be exposed to. Regardless of the W speciation, low W levels decreased the total culture biomass as assessed by DNA concentrations in extracts ( Supplementary Fig. 3), suggesting that microorganisms requiring or using W may play important roles in community productivity, despite the relative abundances of only a few community members being directly affected by the absence of W. The critical W concentration to maintain W. gerlachensis in culture is higher than that required by P. furiosus (at least 1.5 nM but less than 15 nM) 34 . Nevertheless, P. furiosus growth dynamics were not determined over multiple transfers. There may be differences in the W-requiring biochemistry between these two organisms, while the comparison between mixed and pure cultures may also be obscured by inter-species W cycling.
Because the abundance of Caldimicrobium was also affected by W limitation (Fig. 2a and Supplementary Fig. 4), we were unable to exclude the possibility that W. gerlachensis was affected indirectly via Caldimicrobium, potentially due to a mutualistic relationship between the two. Nevertheless, the presence of multiple W-associated genes in the W. gerlachensis MAG, discussed below, suggested that the requirement for W is direct. To probe the genomic potential for W. gerlachensis anaerobic growth on carbohydrates, we annotated a high-quality MAG from a hybrid assembly of short-and long-read sequence data derived from the laboratory cultures (N50 = 86,582 bp, estimated 98.06% completeness and 0.49% contamination, containing both 16S and 23S rRNA genes). This MAG encoded six W-dependent ferredoxin oxidoreductases as well as a complete WO 4 2transmembrane ABC transport system of the Tup family, which corroborated our initial hypothesis that W is required for growth in synthetic medium. W-dependent ferredoxin oxidoreductases are extremely oxygen-sensitive cytoplasmic tungstoenzymes that are involved in sugar and peptide catabolism in a variety of thermophiles. They oxidize various aldehydes, derived from amino acids or sugars, to corresponding carboxylic acids with ferredoxin as the electron acceptor 35 . These enzymes can generally be separated into aldehyde:ferredoxin oxidoreductases (AOR) 24 , glyceraldehyde-3-phosphate:ferredoxin oxidoreductases (GAPOR) 25,36,37 , and formaldehyde:ferredoxin oxidoreductases (FOR) 26 , in addition to tungstoenzymes with unknown function (WOR4) 27 or broad substrate specificity (WOR5) 28 . All characterized enzymes in this family require incorporation of W, which is coordinated by a pyranopterin cofactor, similar to molybdoenzymes 38,39 . Dissolved W is mainly available as the tetrahedral oxyanion WO 4 2in reduced spring waters 40 . Tungstate is taken up by ABC-type transporters containing highly selective binding proteins (TupA, WtpA). Molybdate is transported by other distinct transmembrane proteins (ModABC). Although we found several annotated MoO 4 2cofactor biosynthesis protein-encoding genes within the W. gerlachensis MAG, no Mod genes were present, supporting that multiple cellular processes in W. gerlachensis are dependent on W and not Mo.
To test if the identified genes encoding tungstoenzymes and W transporters were expressed, we performed reverse transcriptasepolymerase chain reaction (RT-PCR) assays specific for these W. gerlachensis transcripts in community RNA. While several of the oxidoreductase genes were transcribed, WO 4 2-ABC-type transporters were only weakly transcribed ( Supplementary Fig. 5). Effects of W on Wolframiiraptor (W. gerlachensis) growth in culture A10. a ASV abundances based on 16S rRNA gene tags from synthetic medium with and without added spring water or W. The 12 most abundant taxa are shown, which consistently made up >98% of the total ASVs. Caldimicrobium and W. gerlachensis ASV counts from triplicate cultures were statistically different between synthetic medium with and without spring water (without W addition) as denoted by the asterisk (ANOVA, *p = 0.07, **p = 0.03, n = 3). ASV counts are reported as raw reads; see Supplementary Data 9 including normalized ASV counts that show identical trends. b Absolute abundance of W. gerlachensis based on 16S rRNA gene qPCR on corn stover incubations. Central marks in boxes indicate the median, with the bottom and top edges as the 25th and 75th percentiles. Error bars denote SD of biological replicates (n = 3). c Abundance of W. gerlachensis 16S rRNA genes measured after each transfer and the resulting growth. Cultures were grown in corn stover and sugar mix in synthetic medium amended with discrete W concentrations (indicated by black arrows). 16S rRNA gene tags retained at the third transfer indicated no ASVs affiliated with W. gerlachensis in all replicates without W ( Supplementary Fig. 4). Source data are provided as a Source Data file.
Transcripts for the WO 4 2--binding portion of the ABC complex (MCF3653660.1) were detected in the sugar mix culture (irrespective of reverse transcriptase), and the transmembrane subunit transcripts were detected in the corn stover culture. Increased synthesis rates of the periplasmic binding proteins over other subunits have previously been observed for ABC-type transporters in Escherichia coli, including MoO 4 2transporters, where the synthesis rate of the periplasmic binding protein exceeded that of the transmembrane portion by at least 100fold 41 . For the putative W-dependent ferredoxin oxidoreductases, the gene encoding protein MCF3653435.1 was transcribed in corn stover and sugar mix cultures, whereas those encoding MCF3653440.1 and MCF3653608.1 were only transcribed in corn stover cultures ( Supplementary Fig. 5). The apparent higher expression of tungstoenzymes in corn stover over sugar mix cultures is consistent with higher sensitivities to W starvation (Fig. 2c). Thus, the expression of W-dependent enzymes varied in enrichments provided with different primary carbon substrates, pointing to the direct involvement of tungstoenzymes in the catabolism of complex carbohydrates in corn stover.
Xylose is the preferred carbohydrate monomer for W. gerlachensis. To investigate carbohydrate metabolism in W. gerlachensis in the absence of an axenic culture, we used a combined CARD-FISH-nanoSIMS approach 42 that allowed us to directly interrogate the metabolism of single cells in the complex community of the enrichment. We amended GBS in situ corn stover enrichments with different 13 C-labeled substrates (amino acids, starch, glucose, xylose, and ribose) to track carbohydrate metabolism, and with 15 N-labeled ammonium to track overall assimilatory activity. Cells from non-FISH-labeled incubations collected after 24 h were significantly enriched in both 15 N and 13 C compared to unlabeled controls (Kruskal-Wallis test, p < 0.001; Fig. 3 and Supplementary Fig. 6), indicating active metabolism. Carbon and nitrogen enrichment were positively correlated (p = 0.003, Supplementary Fig. 6), but the 13 C/ 15 N enrichment varied, suggesting that some cells were metabolically active (labeled with 15 N ammonium) but did not incorporate any of the 13 C-labeled substrates.
The W. gerlachensis population identified with CARD-FISH and subsequently analyzed by nanoSIMS was significantly enriched with 13 C and 15 N in all except the ribose-amended incubations ( Fig. 3 and Supplementary Fig. 6; Kruskal-Wallis test, p < 0.001). W. gerlachensis cells were the most active ( 15 N enriched) in the xylose treatment. Xylose incubations also contained W. gerlachensis cells most enriched in 13 C (median of 15 atom% excess and 100% of cells enriched), consistent with direct assimilation of xylose and a preference for it over the other substrates tested. Median 13 C enrichment of W. gerlachensis cells incubated with labeled glucose, amino acids or starch was less than 1 atom% excess (potentially due to cross-feeding on metabolites derived from these substrates), and no isotope enrichment was detected in 13 C ribose incubations.
A hot spring sediment community composed of 4% Caldarchaeales degraded the xylose polymer xylan and was used to purify a thermostable xylanase enzyme (>70% activity at 70°C 43 ), indicating the participation of this lineage in polysaccharidedegrading consortia. We also found xylose isomerase (EC 5.3.1.5) and multiple xylulose kinase (EC 2.7.1.17) encoding genes in the W. gerlachensis MAG. In light of our NanoSIMS results, these data suggest W. gerlachensis is an important contributor to the anaerobic degradation of lignocellulose by the microbial community at GBS, likely as members of a cellulolytic consortium 17 . The precursor of xylose is likely hemicellulose in corn stover, but other sources might be present in natural hot spring environments, including both allochthonous (e.g., nearby plants) and autochthonous (e.g., biofilm) materials. W-dependent ferredoxin oxidoreductases putatively drive aldehyde metabolism. Next, we identified the potential substrates of the six W-requiring ferredoxin oxidoreductases encoded by the W. gerlachensis MAG, and likely to be compromised by W limitation, employing primary sequence and structural analyses. Over 2600 sequences of known or annotated AOR, FOR, GAPOR, and WOR homologs served as reference sequences to interrogate the W-associated ferredoxin oxidoreductase homologs encoded in the W. gerlachensis MAG. Based on phylogenetic analysis, three homologs were distantly related to wellcharacterized AORs, herein referred to as AOR-like 1, AOR-like 2, and AOR-like 3; one homolog was distantly related to characterized FORs, referred to herein as FOR-like; and one homolog was distantly related to characterized GAPORs, here termed GAPOR-like ( Fig. 4 and Supplementary Figs. 7 and 8). To gain insight into the putative functions of these oxidoreductases, we modeled the protein structure of MAG-encoded oxidoreductase sequences belonging to the genus Wolframiiraptor, along with selected reference sequences, and applied multidimensional scaling on the predicted structures (Fig. 4b). Structural similarities to a characterized AOR from P. furiosus, as quantified based on a sum-of-pairs score using a protein database reference, emerged in the three AOR-like lineages (Cluster I and II, Fig. 4b), supporting the phylogenetic analysis. The other oxidoreductases were structurally related to the GAPOR from Caldicellulosiruptor bescii (Clostridia) 37 (Cluster III, Fig. 4b) and diverse FORs (Cluster IV, Fig. 4b), again congruent with the phylogenetic analysis. With the genomic assessments, coupled with the protein phylogeneticstructural analyses, we further looked for evidence of physiological dependence on W.
W-requiring ferredoxin oxidoreductases are found at the heart of sugar and peptide metabolisms in thermophilic anaerobes 44,45 . While glycolysis in thermophilic bacteria is catalyzed by the conventional glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase, glucose catabolism in several hyperthermophilic archaea differs from that canonical pathway and appears to proceed via a modified Embden-Meyerhof (EM) pathway that involves novel W-dependent enzymes 46 . On one hand, phylogenetic analysis and structural modeling of the homologs represented by MCF3653440.1 in W. gerlachensis predicts an encoded GAPOR (Fig. 4, GAPOR-like lineage). On the other hand, the W. gerlachensis MAG also encodes an annotated glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) and a phosphoglycerate kinase (EC 2.7.2.3) and we thus cannot determine with certainty if pyruvate is originating via the canonical or modified (archaea-typical) EM pathway. Similar to the amino acid catabolism in P. furiosus, W. gerlachensis likely decarboxylates 2-keto acids, such as pyruvate, to aldehydes that are then oxidized by W-dependent ferredoxin oxidoreductases to form carboxylic acids (Fig. 4c) [47][48][49] . The pivotal step of the metabolic inhibition occurs when W-requiring ferredoxin oxidoreductases become insufficiently supplied with the W-cofactor tungstopterin and ferredoxin reduction halts. A ferredoxin-dependent, membrane-bound proton-translocating [NiFe]-hydrogenase (Mbh) encoded by the W. gerlachensis genome would then increasingly lack reduced ferredoxin to trigger tunneling of H + ions across the cell membrane, thereby leading to a decreased transmembrane H + gradient and retarded ATP formation (Fig. 4).
Inactivation of W-dependent ferredoxin oxidoreductases would be highly effective at depriving the cell of energy because (i) the encoded ATPase is predicted to be proton-dependent, based on alignment with V-type ATPases 50 , inducing a proton gradient of 3 ion equivalents across the membrane and (ii) these oxidoreductases can make up a significant proportion of cell biomass. For instance, in Thermococcus strain ES1, these oxidoreductases constitute 1% of the total protein mass 51 and the intracellular W-dependent ferredoxin oxidoreductase concentration in P. furiosus reached 110 µM 44 . Interestingly, the W. gerlachensis MAG revealed a putative ferredoxin-dependent, membrane-bound, and proton-translocating [NiFe] hydrogenase complex of 10 subunits (Mbh-Mrp) including conserved cysteine pairs in the N-and C-terminus of the catalytic subunit MbhL (Supplementary Fig. 9) that ligate the [NiFe] metallocluster 52 , along with several Hyp-type [NiFe] metallocluster assembly proteins. We were not able to find rnf genes 53 in the W. gerlachensis MAG and we hence suspect ferredoxin (as the reducing equivalents produced by AOR) would predominantly be recycled by the Mbh-Mrp. A similar Mbh-Mrp complex has previously been described for P. furiosus 54,55 and is a type-4 [NiFe] energy-conserving hydrogenase 56 that was also detected in other Caldarchaeales genomes 8,12 . W addition into culture media of the marine thermophile Thermotoga maritima resulted in stimulation of hydrogenase activity 57 . The carbon and energy metabolism in that bacterium are similar to that of W. gerlachensis 58 : Formation of pyruvate via the EM pathway, production of organic acids and H 2 and therefore coupling of W enzymatic activity to a hydrogenase and alternative reduction of S 0 to H 2 S by H 2 . Across domains and comprising terrestrial as well as marine habitats, W-associated anaerobic heterotrophic processes are therefore important for thermophilic life.
Distribution of tungstoenzymes across the family Wolframiiraptoraceae. In evaluating the scope of tungstoenzymes in the Caldarchaeales, we recruited and annotated 77 additional high-quality MAGs related to W. gerlachensis that were extracted from metagenomes from geothermal springs in Tengchong, China; Yellowstone National Park, United States; and Taupo, New Zealand, along with a deep-sea hydrothermal mineral deposit from the Eastern Lau Spreading Center in the Southern Pacific. The assignment of these MAGs to the family Wolframiiraptoraceae was defined based on monophyly using conserved marker gene sets and relative evolutionary divergence values. The MAGs could be assigned to four genus-level lineages [including previously recognized AigG4 (syn. EX4484-121, herein Wolframiiraptor), AigG5 (syn. JGI-000106-J15, herein Geocrenenecus), and AigG7 (syn. NZ13-MG1, herein Terraquivivens) 8 ], and 11 species (Fig. 5 and Supplementary Fig. 10), based on relative evolutionary divergence (Supplementary Data 3), average amino acid identity, and average nucleotide identity (Supplementary Data 4).
To assess the potential importance of W within the family Wolframiiraptoraceae, we interrogated the presence of tungstoenzymes within the genomes of these taxa. The three AORlike, FOR-like, and GAPOR-like enzymes were widely distributed within the family, each present in more than half of the Wolframiiraptoraceae genomes ( Fig. 4 and Supplementary Figs. 7 and 8), sparse in other Caldarchaeales, and absent from close relatives in the Nitrososphaeria (Supplementary Fig. 11). In addition, three divergent putative tungstoenzymes were identified in the Geocrenenecus and Terraquivivens MAGs.
All protein-coding sequences annotated as the substratebinding subunits of the Mod (K02020), Tup (K05772), or Wtp (K15495) ABC transporter systems within the family were analyzed to determine the putative substrate specificity of these transporters ( Supplementary Fig. 12a, b). All species of the genera Wolframiiraptor, Terraquivivens, and the sole representative of Benthortus encode homologs of the TupABC transporter. Notably, only a single genome of Geocrenenecus and members of the species T. tengchongensis encoded novel TupA-and/or WtpA-like proteins. The WtpA-like protein has been identified before as a ModA-like homolog in Pyrobaculum species 14,59 . The consensus lack of a MoO 4 2-/WO 4 2transport system in the Geocrenenecus species is not unique, as several completely sequenced genomes from organisms known to utilize MoO 4 2-/ WO 4 2lack any of these annotated ABC transporters 59 .
Scaling up the distribution analysis of MoO 4 2-/WO 4 2transport proteins across entire thermophilic community metagenomes indicated unequal modA, tupA, and wtpA gene frequencies among microbial communities inhabiting springs that span a spectrum of geochemical conditions ( Supplementary   Figs. 13 and 14). While the WO 4 2transporters show a high presence (~70%) in spring waters with temperatures >60°C, wtpA is generally more abundant than tupA and is the most abundant MoO 4 2-/WO 4 2transporter among microbial communities of low-pH (<4) springs.
To better understand how Wolframiiraptoraceae acquired W-dependent enzymes, we inferred associated evolutionary events through phylogenetic analyses, ancestral character state reconstructions (Supplementary File 2), and gene tree/species tree Reference sequences are color-coded. K-means clustering was applied to identify significant data clusters (colored ellipses) based on minimum cluster r 2 . Clusters included AORs from both references and those from representative members of the genus Wolframiiraptor. One sequence (MCF3653595.1) was excluded from the analysis (<300 amino acids). Dotted lines are used to indicate putative groups identified in a and b. All AOR-like lineages grouped together in Clusters I and II, while GAPOR-like sequences grouped in Cluster III and FOR-like sequences grouped in Cluster IV. c Pyruvate, derived from xylose via the pentose phosphate pathway (PPP) and the Embden-Meyerhof pathway (EMP), is stepwise converted to organic acids. Within this conversion, AOR and GAPOR couple aldehyde oxidation with ferredoxin reduction. Electrons from ferredoxin are used by a membrane-bound protontranslocating [NiFe]-hydrogenase (Mbh) to generate a proton motive force. Besides this main pathway, aldehydes could additionally be derived from amino acid metabolism and ferredoxin could also be recycled by an H 2 -consuming sulfide dehydrogenase 89 (Fig. 5c). These analyses revealed a complex evolutionary history characterized by rampant lateral transfer from other Thermoproteota to various Wolframiiraptoraceae lineages. Although it remains difficult to distinguish between phylogenetic noise and true signal for lateral acquisitions in single-gene trees, the presence of one tungstoenzyme (AOR-like 1) in the ancestral node to the Wolframiiraptoraceae was well supported by all analyses. This ancestral enzyme, combined with extensive capture of W-associated enzymes from

Discussion
In the present study, we demonstrate that W is required to culture the anaerobic species W. gerlachensis, the first physiologically characterized cultured member of the Caldarchaeales. The basis of this requirement, as evidenced by poor biomass yields in cultures in synthetic medium without amended W, is most likely W-dependent ferredoxin oxidoreductases involved in the degradation of xylose-derived pyruvate, which becomes non-functional without W. This dependence on W is seemingly a characteristic of several close relatives of W. gerlachensis, with W utilization feasibly representing an ancestral trait within the family and important for the diversification of the Wolframiiraptoraceae. The levels of W required by W. gerlachensis (≥20 nM) are below those that are typically inhibitory to the growth of other microorganisms where W inhibition has been demonstrated 18 . The standard inclusion of this trace element may aid in the cultivation of additional members of the Wolframiiraptoraceae and other novel thermophiles.
It has been suggested that molybdoenzymes did not evolve prior to the widespread oxygenation of the Earth, which allowed for oxidative weathering of sulfide minerals that hosted Mo 60 . In contrast to Mo, W is less affinitive to sulfide complexation and it is possible that the apparent favorability of W over Mo in the Wolframiiraptoraceae is a relic from adaptations to early microbial habitats where W was more bioavailable than Mo. On the early Earth, such potential habitats were more common and extended beyond hot springs into the marine realm. About 2.3 billion years ago, increasingly sulfidic bottom waters of the oceans underwent a strong geochemical shift from ferruginous to euxinic [55][56][57] . Investigation of contemporary euxinic sediments in the Black Sea revealed a critical concentration of~11 µM aqueous sulfide to cause complete Mo fixation from the water column 61 due to reduction to insoluble Mo-sulfide species or transformation to particle-reactive thiomolybdate (MoS 4 2-) 62 . Such sulfide levels based on dissimilatory sulfite/sulfate reduction 63 or volcanically derived sources 63,64 likely prevailed both in terrestrial hot springs and marine basins. The availability of W theoretically could have constituted a regulatory switch on the fermentative degradation of organic matter and could have influenced fluxes of organic acids, which, in turn, were crucial substrates for sulfur reduction and methanogenesis. The selective pressure for W utilization likely dropped with the oxygenation of the Earth and the dispersion of soluble Mo, but the remnants of ancient W-based pathways likely perpetuated in the carbohydrate metabolisms of anaerobic, thermophilic archaea.

Methods
In-situ enrichments and sampling. GBS water used in the preparation of cultivation medium was sampled in 2013, 2016, 2017, and 2018, passed through a 0.2 µm filter, and stored in 20 L plastic containers in the laboratory without temperature control. In situ enrichments on ammonia-fiber explosion-treated corn stover (kindly provided by Bruce Dale, Michigan State University) in GBS were performed as described in Peacock et al. 17 . Samples were taken after approximately six months of incubation on March 29, 2016. At that time, the temperature of the water was 86.0°C and the pH was 7.15, typical for the main source pool of GBS 16 . For sampling, a nylon bag containing corn stover was removed from the conical tube, the bag was cut open with a sterile scissors, and approximately half of the contents was immediately transferred to 10 mL of 0.2 µm filtered GBS water (previously sampled, sparged with N 2 , reduced by the addition of cysteine to 0.05 g/L, and sterilized by autoclaving) in a 25 mL Balch tube under a stream of N 2 gas applied through a 0.2 µm filter. The tube was sealed with a butyl rubber stopper, the headspace was flushed for 3 min with N 2 gas, and the tube was then shaken to disperse the corn stover. Previously prepared anaerobic media (50 mL in 160 mL serum vials sealed with butyl rubber stoppers; see below) were inoculated with 0.2 mL of the corn stover slurry using a sterile, N 2 -flushed needle and syringe. The bottles were transported back to the laboratory at ambient temperature and were transferred to hightemperature incubators within 24 h of sample collection. The remaining in-situ corn stover enrichment was placed in 1.5 mL centrifuge tubes, frozen immediately, and transported back to the laboratory on dry ice, and then stored at -80°C for DNA extraction. O, and 0.024 g NaMoO 4 ·2H 2 O. The "spring water medium" consisted of a 1:1 mix of the above synthetic medium and water collected from GBS as described above. All media contained 0.107 g/L NH 4 Cl. The medium was then sparged with N 2 gas and transferred into serum vials in an anaerobic chamber. The vials were sealed with butyl rubber stoppers and aluminum crimps, the headspace was exchanged with N 2 by three cycles of vacuuming to 585 mm Hg and repressurizing to 15 psi with N 2 , and the vials were sterilized by autoclaving 64 . Prior to inoculation, the following were added to all media at the indicated final concentrations from concentrated, anoxic stocks: 0.025% keratin, 0.002% xyloglucan, 0.02% Na 2 S, 0.02% cysteine, 0.002% yeast extract, 0.0014% NaH 2 PO 4 ·H 2 O, and 1.6 mL/L of a vitamin solution 66 . All stocks were sterilized by autoclaving except yeast extract and vitamins, which were passaged via a 0.2 µm filter. Additions for different media conditions in the initial enrichments were made from sterile, anoxic stocks to final concentrations shown in Supplementary Table 1, except that corn stover (0.02% m/v) was added to individual serum vials in the anaerobic chamber prior to sealing. Follow-up experiments used the same base synthetic or spring water media with modifications of the specific substrates that were excluded or added anoxically. After stable laboratory cultures were obtained, the culture conditions for routine transfer and maintenance used the base spring water medium with 2 mM sodium bicarbonate and either corn Fig. 5 Genomic relatedness (a), phylogenetic placement (b), and distribution of tungstoenzymes (c) in the family Wolframiiraptoraceae. a Average amino acid identity (AAI) among designated type genomes for species-level groups within the family Wolframiiraptoraceae. The same color scheme used in b is used for taxa indicated with numbers. For full ANI and AAI matrices of the respective genera, see Supplementary Data 4. b A maximum-likelihood phylogeny was inferred from the concatenated, partitioned sequence alignment of 122 conserved archaeal marker sequences (ar122). Appropriate evolutionary models were applied for each partition. Branch support for the phylogeny was inferred from 1000 pseudoreplicates, using traditional bootstrapping and supported branches are indicated with filled circles at nodes. Species-level groups, as supported by phylogenetics, average nucleotide identity (ANI), and relative evolutionary divergence are indicated with different colors, with species belonging to the same genus indicated in different shades of the same color. Taxon names in bold and indicated with a star are the proposed nomenclatural types for the taxa. Gray taxon names represent medium-quality MAGs that are publicly available. c Summary of gene tree/species tree reconciliation analyses with predicted gains, transfers, and losses, for W-associated enzymes (ABC-type transporter A subunits and W-dependent oxidoreductases) within the family. Hypothesized evolutionary paths for the genes of interest are indicated in the colors used in the dot matrix on the right of the cladogram and numbered according to the dot matrix order. The consensus presence or absence profiles for species with multiple genome sequences available were determined by data as indicated for the majority of genomes in the species. Where genes encoding these proteins are present in some genomes within a taxon, colored dots are partially filled, while fully filled dots in the dot matrix indicate the presence in the majority of the genomes belonging to that taxon. For full ABC-type transporter phylogeny, including KOs stover (0.02% m/v) or a "sugar mix" (0.01% m/v each of glucose, xylose, mannose, and D-and L-arabinose) for major carbon sources. Laboratory cultures were kept at 80°C and initially transferred to fresh media (1/100 volume) every 2 weeks for the first two transfers after inoculation with in situ enrichments and then every 3 weeks thereafter.
Quantification of W in spring water and media by ICP-MS and ICP-OES. W levels in laboratory media and filtered GBS spring water were measured using ICP-MS after the addition of nitric acid (OPTIMA grade, Fisher Chemical, Fair Lawn, NJ, USA) to 1% final concentration at Huffman Hazen Laboratories (Golden, CO, USA). W levels in filtered GBS spring water were also measured using inductively coupled plasma optical emission spectroscopy (ICP-OES) with an Agilent 5110 ICP-OES (Santa Clara, CA, USA) and dilutions of a W calibration standard (PLW9-2Y, SPEX CertiPrep, Metuchen, NJ, USA). Nitric acid was added to samples and standards to a final concentration of 1% before analysis, and optical emission at 207.91 nm was used for W quantification. Broad trace element analysis by ICP-MS on GBS spring water and media was performed on samples prepared as described above at the Analytical Chemistry Laboratory of New Mexico Bureau of Geology and Mineral Resources, New Mexico Institute of Mining and Technology.
16S rRNA gene tag sequencing. 16S rRNA gene tag amplification and sequencing on initial enrichment cultures was performed essentially as described 67 . A modified forward primer 515 (5′ GTGYCAGCMGCCGCGGTAA) with a Y instead of a C at the fourth position from the 5′ end was used to increase coverage of Archaea, and a corresponding change was made to the SeqRead1 primer. Sequencing (2 × 250 bp) was performed on an Illumina iSeq sequencer using the V2 reagent kit. Additional 16S rRNA gene tag sequencing on laboratory cultures tested with different levels of W was performed as described 68 at Argonne National Laboratories.
Genomic DNA and RNA extraction. For the laboratory culture, biomass (from 10 to 40 mL culture) was harvested by centrifugation at 10 min at 9300 g and 4°C in 50 mL conical tubes. Resulting pellets were resuspended in 1 mL of supernatant, transferred to 1.5 mL tubes, and centrifuged at 16,100 g for 5 min at 4°C. The supernatant was removed and the pellets were stored at -80°C until nucleic acid extraction. DNA was extracted using the Fast DNA SPIN Kit for Soil (MP Biomedicals) as described following the manufacturer's recommendations, except that homogenization by bead-beating was performed twice for 30 s at a speed setting of 4.5 m/s. DNA was eluted in 75-100 µL of DES solution and further diluted 10-fold in 10 mM trishydroxyaminomethane pH 8. To obtain long fragments of DNA for Oxford Nanopore sequencing, DNA was extracted using the cetyltrimethyl ammonium bromide protocol of the Joint Genome Institute 66 . RNA was extracted using the FastRNA Spin Kit for Soil (MP Biomedicals) and the High Pure RNA Isolation Kit (Roche) as described in Hamilton et al. 69 , followed by an additional DNase treatment using the TURBO DNase kit (Ambion) according to the manufacturer's instructions. For all other information regarding metagenomic DNA extraction, please refer to Supplementary Note 1.
Quantitative PCR. Assays were performed in 96-well plates (Applied Biosystems) in a StepOnePlus Quantitative Real-Time PCR machine (Applied Biosystems) using PowerUp SYBR Green Master Mix (Applied Biosystems). Cycling conditions for AigG4-specific primers (Supplementary Table 2) were as follows: initial denaturation for 15 min at 95°C followed by 45 cycles of denaturation (20 s at 95°C), annealing (25 s at 64°C), and extension (50 s at 72°C). Specificity of qPCR primers was verified by melt curve analysis. Using a plasmid with a fragment of the AigG4 16S SSU rDNA sequence as a standard (SSW_L4_C03; ref. 23 ), an AigG4 standard curve was generated over 6 orders of magnitude from 5.0 × 10 2 to 5.0 × 10 8 (r 2 = 0.98-1.00). The reported template abundances are the average and standard deviation of qPCR assays performed in triplicate. Quantification of total bacterial and archaeal 16S rRNA genes was performed similarly using primers 806r and 515f with the modification described above for 16S rRNA gene tag sequencing (Supplementary Table 2), and with an annealing temperature of 55°C.
Reverse transcriptase PCR. RNA was converted to cDNA with random hexamer primers using the Verso cDNA kit (ThermoScientific), including control reactions without added reverse transcriptase. cDNA was amplified with primers targeting AigG4 predicted W-associated genes and AigG4 16S rRNA (Supplementary  Table 3) in the Axygen® Maxygene™ II Thermal Cycler as follows: at 95°C for 3 min followed by 35 cycles of denaturation (30 s at 95°C), annealing (30 s at annealing temperature appropriate for specific primer set), and extension (72°C for 5 min) and finally, 72°C for 1 min. Primers targeting AigG4 predicted W-associated enzymes were designed using Primer3 65 . DNA was quantified using the Qubit 4.0 with HS DNA reagent kit.
Stable isotope labeling. Stable isotope labeling was performed on-site at GBS in July 2017 using in situ corn stover enrichments prepared and incubated as described above but using 5 g packets of corn stover incubated at the GBS "C" site (outflow) 16 for 34 days. Two packets (approximately 10 g) of freshly harvested corn stover enrichment were added to 180 mL of anaerobic, autoclaved GBS water in a sealed 500 mL bottle with an N 2 headspace and shaken to form a slurry. Samples of this slurry (2 mL) were then added via syringe to sealed 60 mL amber serum vials containing 8 mL of anaerobic, autoclaved GBS water with an N 2 headspace, generating 10 mL microcosms. Various individual 13 C-labeled substrates (Cambridge Isotope Laboratories) were then added from concentrated, anaerobic stocks to the microcosms at 0.001% (mass/volume) final concentration: algal amino acids, algal starch, glucose, xylose, ribose, or an equal-mass mixture of acetate, propionate, and butyrate; a control with no label added was also performed and did not yield enriched cells. In addition, 0.5 mM 15 N-NH 4 Cl was added to all microcosms. Preparation of microcosms was done in a hot water bath at 75°C to prevent excessive cooling. Unlabeled controls were processed as described below immediately after preparation. Sealed microcosms with labeled substrates were incubated at 75°C for 4 or 24 h. After incubation, microcosm vials were immersed in ice water to cool, shaken vigorously for 30 s, opened, and decanted into 15 mL conical tubes. Tubes were centrifuged for 30 s at 11 g in a clinical centrifuge to pellet large fragments of corn stover. Supernatant (4.5 mL) was then split between three 1.5 mL tubes and centrifuged for 5 min at 16,100 g to pellet cells. Pellets from the three tubes were pooled in 0.25 mL of 1×PBS, and 0.5 mL of 3% paraformaldehyde (PFA) was added and mixed. Samples were fixed for 1 h on ice. After fixation, tubes were centrifuged for 5 min at 16,100 g. Resulting pellets were washed twice with 1×PBS, resuspended in 200 µL of 50% ethanol, and stored on ice (in the field) and then at -20°C in the laboratory. Prior to performing FISH, Nycodenz density cushion centrifugation 70 was used to separate cells from higher density particles in the samples. Cells harvested from the aqueous-Nycodenz interface were pelleted by centrifugation, washed twice in water, and resuspended in 50% ethanol.
CARD-FISH. Detection of W. gerlachensis (AigG4) cells was performed using CARD-FISH using a horseradish peroxidase-labeled probe 5′ CRGAAAGGCCTT CAACCTGT (Biomers.net, Germany). This probe is specific to Aigarchaeota Group 4 and Group 5, and was designed based on multiple sequence alignment of 16S rRNA genes of Aigarchaeota and other Archaea and checked using the Probe Match function of the Ribosomal Database Project 16 . Optimization of formamide concentrations used during hybridization was performed using the Clone-FISH technique 70 with the AigG4 16S rRNA gene sequence SSW_L4_C03 (EU635908) 23 expressed from the T7 promoter on plasmid pET23(+) in E. coli strain JM109(DE3). Formamide concentrations from 0 to 50% were tested in 10% increments. The highest formamide concentration that gave strong signal (20%) was then used to test for signal in the E. coli strain without induction of expression of the AigG4 16S rRNA as well as Staphylococcus aureus and Bacillus subtilis as negative controls. CARD-FISH was performed as previously described 71 with minor modifications. Briefly, cells were fixed using a 1% PFA solution for 1 h, followed by storage at -20°C until use. The protocol was performed on fixed cells spotted and air dried on custom synthesized 14-well indium tin oxide (ITO)-coated slides rather than using filters. Cells were permeabilized by treatment with 10 mg/ mL lysozyme (for Clone-FISH in E. coli and negative controls) or 1 µg/mL proteinase K (for samples with AigG4) in 50 mM EDTA and 100 mM Tris (pH 8) for 60 min at 37°C, endogenous peroxidases were inactivated by treatment with 0.01 M HCl for 15 min at ambient temperature (~25°C), and dehydrated using 96% EtOH. Hybridization was performed at 20% formamide for 16 h at 46°C, followed by washing at 48°C, CARD using tyramide labeled with Alexafluor 555 (Invitrogen), and counterstaining by incubation in ice-cold 1 ug/mL DAPI for 10 s. Cells were visualized with a Leica DM5500B microscope with a ×100 magnification dry immersion objective. Fluorescence and brightfield images were collected at diverse locations with FISH-positive cells and the X-Y location of those images was noted, as well as fiducial locations to enable navigation in the nanoSIMS.
NanoSIMS. ITO-coated slides were cut with a diamond saw in order to fit on metal sample holders and analyzed on a CAMECA nanoSIMS 50 at Lawrence Livermore National Laboratory. The charge-coupled device camera was used to find fiducial locations and locations of the FISH-positive cells using X-Y coordinates and the real-time imaging unit. The primary 133 Cs + ion beam was set to 1.5 pA, corresponding to an approximately 100 nm diameter beam diameter at 16 keV. Rastering was performed over 20 × 20 μm analysis areas with a dwell time of 1 ms pixel -1 for 19-30 scans (cycles) and generated images containing 256 × 256 pixels. Sputtering equilibrium at each analysis area was achieved with an initial beam current of 90 pA to a depth of~10 nm, thus ensuring analysis of intracellular isotopic material but without sputtering through the ITO coating. After tuning the secondary ion mass spectrometer for mass resolving power of~7000, secondary electron images and quantitative secondary ion images were simultaneously collected for 12 C 2 -, 13 C 12 C -, 12 C 14 N -, and 12 C 15 Non individual electron multipliers in pulse counting mode. All nanoSIMS datasets from two independent experimental runs were initially processed using L'Image (http://limagesoftware.net, August 2021) to perform deadtime and image shift correction of ion image data before creating 13 C 12 C/ 12 C 2 and 12 C 14 N/ 12 C 15 N ratio images, which reflected the level of 13 C and 15 N incorporation into biomass. Regions of interest for isotopic ratio quantification were drawn manually around each cell. A cell was considered isotopically enriched, and therefore anabolically active, if the isotopic composition exceeded three times the standard deviation of the mean isotopic composition of unlabeled (non-AigG4) cells 72 .
Metagenome sequencing, assembly, and binning. For the AigG4 cultures established from the in-situ enrichment at GBS, extracted DNA were sequenced using the Illumina MiSeq platform (2 × 250 bp), along with Oxford Nanopore sequencing using a MinION device. All extracted metagenomic DNA from geothermal springs from Tengchong, China, and Yellowstone National Park, USA, and the marine hydrothermal vent in the Western Pacific were sequenced on the Illumina NovaSeq, HiSeq3000, or HiSeq4000 platforms, in a paired-end or singleread configuration (Supplementary Note 2). Quality control of sequencing reads are outlined in the Supplementary Notes. Sequencing data were assembled and metagenomes were binned using several software packages (Supplementary Note 2). All MAGs were subjected to quality checks with CheckM v. 1.0.18-1.1.3 73 and classification with GTDB Toolkit v. 1.4.1 74 . This was followed by gene calling using Prodigal v. 2.6.2 75 and annotation of all MAGs using eggNOG-mapper v. 2 76 , and gene calling and annotation with Rapid Annotation using Subsystem Technology 77 in the case of the hybrid assembly for W. gerlachensis.
Phylogenomic analyses. All MAGs classified as belonging to the family NZ13-MG1 in the order Caldarchaeales were used for the construction of a species tree. As reference sequences, genomes for members of the Nitrososphaerales (syn. Thaumarchaeota), along with publicly available genomes for the Caldarchaeales (syn. Aigarchaeota) were included in the taxon set. The 122 conserved archaeal marker sequences in the ar122 dataset were identified from the genomes of these taxa using the GTDB Toolkit v. 1.4.1 74 . Aligned markers were subjected to testing for appropriate evolutionary models using ProtTest v. 3.4 78 , followed by concatenation and partitioning with FASconCAT-G v. 1.0.4 79 . This partitioned, concatenated matrix was subjected to maximum-likelihood tree reconstruction with RAxML v. 8.2.12 80 , with the appropriate evolutionary model applied to each partition, and branch support inferred from 1000 bootstrap pseudoreplicates. For description on the phylogenomic analysis for the domain Archaea, please see Supplementary Notes 3-6.
Phylogenetic analyses of genes. For phylogenetic confirmation of function of the predicted genes related to physiology, and specifically tungstoenzymes, all Wolframiiraptoraceae sequences annotated as belonging to the KEGG Orthology (KO) numbers of interest were used to infer phylogenetic trees to place the identified sequences into evolutionary context within the protein families. A detailed description of these analyses is provided in the Supplementary notes (see also Supplementary Data 7 and 8).
Gene tree/species tree reconciliation and ancestral character state reconstruction. For reconciliation analyses, a species tree was inferred from the domainlevel phylogenomic tree with Bacteria specified as the outgroup. Similarly, subtrees for all W-associated homologs were inferred from the maximum-likelihood phylogenetic analyses of single-gene trees (see Supplementary materials for full description of single-gene tree construction). The inferred gene trees were reconciled with the species tree for the family ecceTERA v. 1.2.4 81 , with default reconciliation penalties, partially ultrametric trees specified, and the median reconciliation graphs printed as sylvx reconciliations. Resulting sylvx reconciliations were viewed in SylvX v. 42 82 , and median symmetrical reconciliations were summarized and edited in Inkscape v. 0.92.4.
A cladogram inferred from the species tree constructed from the genomes was used as the basis for performing reconstruction of ancestral character states for the family, with a particular focus on genes relating to physiology and W utilization. As the presence or absence of genes within single MAGs can be unreliable, a majorityrule consensus approach was employed for all genomes of the respective species. This entailed using all eggNOG annotated genomes, and summarizing the genome content based on KO numbers for each genome. The absence (0), presence (1), or presence in multiple copies (≥2) for each species was determined based on all genomes belonging to the species. These consensus presence/absence profiles for the species were then used to reconstruct all potential evolutionary events (gene birth, HGT, duplication, or loss) by optimizing rates and inferring the history of gene families by posterior probabilities, as implemented in COUNT 83 . The COUNT sessions for the full genome analysis and the tungstoenzyme-specific analysis are available as Supplementary Data 6.
Protein structure modeling. W-dependent oxidoreductase sequences were submitted to the iterative threading assembly refinement (I-TASSER) server 84 to generate models of oxidoreductase homologs. Models were selected based on C-score 85 . All Wolframiiraptor W-dependent oxidoreductases reached at least a C-score of 1.3. Structural comparison of all-against-all was done with DaliLite v5 based on PDB coordinates of the models 86,87 . The first two eigenvectors of the output were used for the multidimensional-scaling and K-means clustering 88 applied in JMP Pro (Version 13.1.0, SAS Institute Inc.).
Reporting summary. Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Data availability
Sequence data for nomenclatural types and some non-type genomes were deposited on GenBank under accession numbers listed in Supplementary Data 3, and all other genomes were submitted to eLMSG and/or IMG under under Bioproject nos. PRJNA495050, PRJNA791658, PRJNA807863, PRJNA381623, and PRJNA780009. For nomenclatural types, raw sequence reads were submitted to the Sequence Read Archive (SRA) under the SRA run accessions listed in Supplementary Data 3. All other data are contained within the manuscript and supplements/source data files. Source Data are provided with this paper.