Diversity of Mixotrophic Neutrophilic Thiosulfate- and Iron-Oxidizing Bacteria from Deep-Sea Hydrothermal Vents

At deep-sea hydrothermal vents, sulfur oxidation and iron oxidation are of the highest importance to microbial metabolisms, which are thought to contribute mainly in chemolithoautotrophic groups. In this study, 17 mixotrophic neutrophilic thiosulfate- and iron-oxidizing bacteria were isolated from hydrothermal fields on the Carlsberg Ridge in the Indian Ocean, nine to the γ-proteobacteria (Halomonas (4), Pseudomonas (2), Marinobacter (2), and Rheinheimera (1)), seven to the α-proteobacteria (Thalassospira, Qipengyuania, Salipiger, Seohaeicola, Martelella, Citromicrobium, and Aurantimonas), and one to the Actinobacteria (Agromyces), as determined by their 16S rRNA and genome sequences. The physiological characterization of these isolates revealed wide versatility in electron donors (Fe(II) and Mn(II), or thiosulfate) and a variety of lifestyles as lithotrophic or heterotrophic, microaerobic, or anaerobic. As a representative strain, Pseudomonas sp. IOP_13 showed its autotrophic gowth from 105 cells/ml to 107 cells/ml;carbon dioxide fixation capacity with the δ13CVPDB in the biomass increased from −27.42‰ to 3460.06‰; the thiosulfate-oxidizing ability with produced SO42− increased from 60 mg/L to 287 mg/L; and the iron-oxidizing ability with Fe(II) decreased from 10 mM to 5.2 mM. In addition, iron-oxide crust formed outside the cells. Gene coding for energy metabolism involved in possible iron, manganese, and sulfur oxidation, and denitrification was identified by their genome analysis. This study sheds light on the function of the mixotrophic microbial community in the iron/manganese/sulfur cycles and the carbon fixation of the hydrothermal fields.


Introduction
Iron and sulfur are elements widely present in the earth. The sources of iron and sulfur in the ocean include dust, coastal and shallow sediments, sea ice, and hydrothermal fluids [1]. Due to the perennial lack of light, the hydrothermal fluid ejected from the deep-sea hydrothermal vent contains a large amount of low-valent iron, manganese, reducing sulfide, methane, hydrogen, and other reducing compounds, which can be used as an electron donor for chemoautotrophic bacteria [2]. Additionally, heterotrophic γproteobacteria and α-proteobacteria were occasionally reported to have lithotrophic iron-, manganese-, and sulfur-oxidation capacities in some environments. Edwards et al. first reported that strains belonging to γ-proteobacteria (Alcanivorax sp., Halomonas sp., Marinobacter sp., and Pseudomonas sp.), α-proteobacteria (Aurantimonas sp., Nitratireductor sp. Stappia sp., and Hyphomonas sp.), and Actinobacteria (Microbacterium sp.) isolated from the eastern flank of Juan de Fuca Ridge off the coast of the Pacific Northwest were identified to have iron-oxidation and nitrate-reduction functions [3,4]. Representatives of six genera (Metallogenium, Leptothrix, Siderocapsa, Naumaniella, Bacillus, and Pseudomonas) in the phyla of Proteobacteria and firmicutes were also proven to participate in the oxidation of Fe(II) and Mn(II) in the bottom sediments of Lake Baikal in Russia [5]. Elemental iron and sulfur

Determination of Iron-Oxidation Capacity
The growths of isolated strains with a solid electron donor in the gradient tubes were tested. The varieties of electron donors include zero-valent iron, FeS, FeCO 3 , basalt, and iron-manganese nodules. FeCO 3 and FeS were prepared as described by Hallbeck et al. [10] and Hanert et al. [11]. All reagents were chemically pure (CP) grade unless otherwise indicated. The basalt, pyrite, and iron-manganese nodules used are natural minerals from deep-sea environments, which were ground and sterilized for later use. The semisolid media with different electron donors were prepared separately. The strains were inoculated into the medium and cultured at 28 • C for up to 2 months.
Aerobic and anaerobic growths were also tested in aqueous media with oxygen or nitrate as electron acceptors. The preparation of stock solutions of FeCl 2 was prepared as described by Emerson et al. [8]. The artificial seawater (ASW) in the iron oxidationnitrate-reduction medium included 28.13 g NaCl, 0.77 g KCl, 1.60 g CaCl 2 ·2H 2 O, 4.80 g MgCl 2 ·6H 2 O, 0.11 g NaHCO 3 , 3.50 g MgSO 4 ·7H 2 O, 0.001 g resazurin, and 1000 mL distilled water. The medium included 5 mM nitrate as the electron acceptor and was supplemented by 1/10,000 of yeast extract (Oxoid, Basingstoke, UK). Serum bottles with 50 mL volume were filled anoxically with 20 mL medium and were sealed with butyl rubber stoppers. The liquid in each serum bottle is gassed with a filter-sterilized N 2 :CO 2 (80:20 v/v) gas mix for a minimum of 3 min. After autoclaving, the medium was supplemented by 10 mM NaHCO 3 , 1 mL/L Wolfe's trace mineral solution, 1 mL/L vitamin solution, and 10 mM FeCl 2 ·4H 2 O. The supplemented solution was sterilized by filtration through 0.22 µm Millipore filters. The anaerobic iron-oxidizing medium contained nitrate as the electron acceptor and FeCl 2 ·4H 2 O as the energy source and reducing agents. The concentrations of nitrite produced divalent iron, and the total iron in the supernatant and that in the precipitate in the medium were measured.

Determination of Manganese-Oxidation Capacity
The growth in the K medium with 1 mM MnCl 2 was added to test the ability of manganese oxidation. Per 1000 mL of distilled water, the medium contained 27.5 g NaCl, 0.05 g K 2 HPO 4 , 1.40 g CaCl 2 ·2H 2 O, 1.00 g NH 4 Cl, 5.38 g MgCl 2 , 0.72 g KCl, 6.78 g MgSO 4 ·7H 2 O, 2.0 g Peptone, 0.5 g yeast extract, and 4.766 g HEPES, and it was adjusted to pH 7.6 with NaOH. After autoclaving, the medium was supplemented by 1 mL sterilized MnCl 2 (1 M) [12]. The strains were inoculated into K agar medium and cultivated for up to 20 days at 28 • C. The presence of Mn oxides around colonies was confirmed by using the leucoberbelin blue (LBB) assay [13].

Determination of Thiosulfate-Oxidizing Capacity
Growth in the autotrophic sulfur-oxidizing medium with sodium thiosulfate as the sole electron donor and sodium bicarbonate as the sole carbon source was observed, which was modified according to Lyu et al. [14]. The artificial seawater (ASW) in the SOB medium included 30.00 g NaCl, 0.14 g K 2 HPO 4 , 0.14 g CaCl 2 ·2H 2 O, 0.25 g NH 4 Cl, 4.18 g MgCl 2 ·6H 2 O, 0.33 g KCl, 0.005 g NiCl 2 ·6H 2 O, and 1000 mL distilled water. After autoclav-ing, the medium was supplemented by sterilized 10 mL trace mineral solution, 10 mL/L vitamin solution, 12.5 mL NaHCO 3 (8%), and 10 mL Na 2 S 2 O 3 (1 M). Before inoculation, the cells were washed three times with sterilized seawater to exclude interference from organic carbon sources and then transferred to the autotrophic media three times. The strains were inoculated into the medium and cultured at 28 • C for 10 days, and the pH, thiosulfate, and sulfate concentrations were measured.

Determination of Carbon-Fixation Capacity
Carbon-fixation experiments were performed using autotrophic sulfur-oxidation medium with 10 mM NaH 13 CO 3 as sole carbon source and sodium thiosulfate as sole energy source. Before inoculation, the organisms were washed three times with sterilized seawater to exclude interference from organic carbon sources and transferred three times. The strains were inoculated into the medium and cultured at 28 • C for 11 days. The pellets were lyophilized for 24 h and determined by Stable Isotope Ratio Mass Spectrometer (IRMS) (measuring accuracy = ±0.2‰; DeltaVAdvantage; Bremen, Germany). The measured method for δ13C‰ was referred to in Orcutt et al. [15]. The growth curve was determined by measuring the OD value of the culture medium, and the absorbance was tested at 600 nm by using a spectrophotometer (Varioskan LUX, Thermo Scientific, Waltham, MA, USA).

Measurements of Fe(II), Fe(III), Sulfate, and Nitrate
Ferrous iron was photometrically quantified with ferrozine after dilution in 1 M HCl [16]. Samples for total Fe analysis were reduced to the ferrous state with 200 mM hydroxylamine for 22-24 h [16].
Sulfate was detected by the barium-ion indication method [17] and the chromatography detection method by using an ion chromatograph (ICS1100, Thermo Scientific, Waltham, MA, USA) [18]. Nitrate and nitrite were detected via the sulfanilamide method [19].

Fluorescence Microscopy and Scanning Electron Microscopy/Energy Dispersive X-ray Spectroscopy (SEM/EDS)
The cells were observed using the LIVE/DEAD BacLight Bacterial Viability Kit (Invitrogen, Carlsbad, CA, USA), which contained SYTO 9 and propidium iodide [20], whose excitation/emission wavelengths were respectively set at 480/500 nm and 490/635 nm to perform dual-channel imaging for green and red fluorescence and further counted using a fluorescence microscope (Eclipse 80i, Nikon, Tokyo, Japan). The pellets were gently mounted on a 0.2 µm pore-size polycarbonate filter and air-dried to further examine them by using a FEI/Quorum PP3000T field-emission instrument (Quorum, Laughton, UK) and to analyze the elements by energy dispersive spectroscopy (EDS).

Nucleotide Sequence Accession Numbers for Strains
GenBank accession numbers of the 16S rRNA gene and genome for strains were deposited. They are shown in Table 1.

Phylogenetic Affiliations
Previous studies showed that most Fe-oxidizing bacteria (FeOB) belong to the bacterial phylum Proteobacteria, including the α-, β-, γ-, δ-, and ζclasses [35]. Figure 1 shows the phylogenetic relationships among 17 strains in this study relative to other known representative FeOB. All the isolates in this study fell mainly within the αand γclasses of the Proteobacteria, except for strain IOP_2, belonging to the phylum Actinobacteria. Strains IOP_29 and IOP_41 were affiliated with Marinobacter (M. adhaerens HP15 and M. shengliensis). Some Marinobacter strains were reported to have neutrophilic iron-oxidizing capabilities from the in situ and lab enrichments of basalts, olivine minerals, etc. near hydrothermal vents, the subseafloor, and iron mine [4,6,[36][37][38]; sulfur-oxidizing capabilities from marine sediments [37,39]; and Mn(II)-oxidizing capabilities from submarine basalts at Loihi seamount off the southeast coast of the island of Hawaii [40]. Four strains, namely IOP_6, IOP_14, IOP_19, and IOP_31, belong to the genus Halomonas by 16s rRNA gene analysis but with low ANI and AAI values (Supplementary Table S1). Halomonas spp. have been frequently identified as Mn(II)-oxidizing and Fe(II)-oxidizing bacteria from basalt in the Juan de Fuca Ridge flank, the volcanic Loihi seamount [36,40,41], the hydrothermal fields in Juan de Fuca Ridge flank, and [36,42] the acid mine drainage (AMD) environments in Southwest China [43]. Some Halomonas strains isolated from hydrothermal fluids and the sediment of hydrothermal field in the East Pacific Rise also showed sulfur-oxidizing capacity [44]. Strains IOP_13 and IOP_25 belong to genus Pseudomonas, which also has been frequently reported as iron-oxidizing bacteria. As described in Sudek et al. [7], Pseudomonas spp. had iron-oxidizing and Mn(II)-oxidizing capacities under microaerobic conditions from a volcanic seamount in the Juan de Fuca Ridge flank. Pseudomonas sp. FE13-26 extracted from sludge was also reported to efficiently oxide Fe(II) with extracellular enzyme ferroxidase [45]. Pseudomonas sp. LOB-2 was reported to have Mn(II)-oxidizing capacity isolated from submarine basalts at the Loihi seamount [40].  Additionally, seven isolates were affiliated to genera Thalassospira, Qipengyuania, Salipiger, Seohaeicola, Martelella, Citromicrobium, and Aurantimonas in the class α-proteobacteria ( Figure 1, Table 2). Most of the iron-oxidizing α-proteobacteria were phototrophs, with the exception of Paracoccus ferrooxidans [46,47], which was a nitrate-dependent bacterium. Moreover, α-proteobacteria (Aurantimonas sp., Nitratireductor sp., Stappia sp., and Hyphomonas sp.) isolated from the eastern flank of Juan de Fuca Ridge were identified to have iron-oxidation and nitrate-reduction functions [4]. The genera Thalassospira and Martelella isolated from hydrothermal sulfides of the South Atlantic were previously also characterized to exhibit a sulfur-oxidizing ability [48]. Those results and our data all proved that some strains in α-proteobacteria had the ability of iron and/or sulfur oxidation to adapt to environmental conditions in deep-sea hydrothermal vents, which had not been notified before.

Growth Test with Different Electron Donors and Acceptors
The growth tests with different electron donors and acceptors in gradient tubes and liquid medium under the aerobic, microaerobic, and anaerobic conditions for all strains are summarized in Table 2.
Under anaerobic conditions, all nine γ-proteobacteria and four of seven strains in α-proteobacteria (Salipiger, Seohaeicola, Martelella, and Citromicrobium) could anaerobically grow with oxidizing FeCl 2 and reducing nitrate. The growth of the cells, nitrite accumulation, and yellow-green precipitate after 40 days of all strains in anaerobic iron-oxidation media were examined. The results showed that under light and fluorescent microscopy, all the cells were observed to be encrusted with Fe(III) minerals, and some cells became dead with red fluorescent (Figure 2). Some isolates (IOP_6, IOP_14, IOP_19, IOP_31 in genus Halomonas; IOP_13 and IOP_25 in genus Pseudomonas; IOP_29, IOP_41 in genus Marinobacter; IOP_21 in genus Rheinheimera; IOP_16 in genus Salipiger; IOP_23 in genus Seohaeicola; IOP_24 in genus Martelella; and IOP_28 in genus Citromicrobium) produced yellow trivalent iron-oxide precipitates in the bottles during the process of cultivation. Nitrite is usually produced an intermediate product produced by bacteria using nitrate as an electron acceptor for anaerobic respiration. The accumulation of nitrite in those cultures was determined to be significantly higher (12.15 to 82.03 µM) than that in the negative control. This demonstrated that these strains use nitrate as an electron acceptor for anaerobic respiration. The nitrate reduction, nitrite formation, and Fe(II) oxidation were not observed in the noninoculated control medium. Strain Pseudomonas sp. IOP_13 produced significantly more nitrite than other strains and produced an orange trivalent iron-oxide precipitate after 10 days of growth (Supplementary Figure S3). The microscopy and EDS analysis of the strain Pseudomonas sp. IOP_13 showed that the cells were encrusted with minerals containing iron ( Figure 2). The aqueous Fe(II) concentration in the culture decreased from 10 mM to 5.20 mM with dissolved Fe(III), which ranged from 0 mM to 2.2 mM after 40 days of growth ( Figure 3A). The orange iron-oxide precipitate contained absorbed 20.63 mM/g Fe(II) and 314.58 mM/g Fe(III) ( Figure 3B). copy and EDS analysis of the strain Pseudomonas sp. IOP_13 showed t encrusted with minerals containing iron (Figure 2). The aqueous Fe(II the culture decreased from 10 mM to 5.20 mM with dissolved Fe(III), w 0 mM to 2.2 mM after 40 days of growth ( Figure 3A). The orange iron contained absorbed 20.63 mM/g Fe(II) and 314.58 mM/g Fe(III) (Figure

Manganese-Oxidizing Capacity
Manganese-oxidizing bacteria can oxidize manganese chloride to manganese dioxide and produce dark-brown colonies on manganese-oxidizing plates. All isolates were cultured in a manganese-oxidation medium, and LBB assay was used on colonies cultured for 20 days. The redox stain (LBB) turned blue in the presence of the accumulation and conversion of manganese by strains Pseudomonas spp. IOP_13, IOP_25, and Salipiger sp. IOP_16.
Strain IOP_16 showed the capacity for Mn(II) oxidation; the closest organism to this strain was the manganese-oxidizing bacterium Salipiger manganoxidans VSW210, isolated from a shallow-water hydrothermal vent in Espalamaca (Azores) [50]. Two strains in the genus Pseudomonas (IOP_13 and IOP_25) that were capable of oxidizing Mn(II) were also identified. Supplementary Figure S4B showed the formation of a blue color on the colonies of strain IOP_13 after LBB staining. Orange halos around the colonies showed its siderphore-producing ability (Supplementary Figure S4A). Some Pseudomonas spp. were reported to have a potential of Mn(II)-oxidizing capacity. Kepkay and Nealson (1987) reported the occurrence of the growth of marine bacteria Pseudomonas sp. S-36, both mixotrophically on succinate or bicarbonate with Mn(II), in Mn-limited chemostats [51]. Mn(II)-oxidizing bacterium Pseudomonas putida GB-1 from a freshwater environment was also identified [52][53][54][55].

Manganese-Oxidizing Capacity
Manganese-oxidizing bacteria can oxidize manganese chloride to manganese dioxide and produce dark-brown colonies on manganese-oxidizing plates. All isolates were cultured in a manganese-oxidation medium, and LBB assay was used on colonies cultured for 20 days. The redox stain (LBB) turned blue in the presence of the accumulation and conversion of manganese by strains Pseudomonas spp. IOP_13, IOP_25, and Salipiger sp. IOP_16.
Strain IOP_16 showed the capacity for Mn(II) oxidation; the closest organism to this strain was the manganese-oxidizing bacterium Salipiger manganoxidans VSW210, isolated from a shallow-water hydrothermal vent in Espalamaca (Azores) [50]. Two strains in the genus Pseudomonas (IOP_13 and IOP_25) that were capable of oxidizing Mn(II) were also identified. Supplementary Figure S4B showed the formation of a blue color on the colonies of strain IOP_13 after LBB staining. Orange halos around the colonies showed its siderphore-producing ability (Supplementary Figure S4A). Some Pseudomonas spp. were reported to have a potential of Mn(II)-oxidizing capacity. Kepkay and Nealson (1987) reported the occurrence of the growth of marine bacteria Pseudomonas sp. S-36, both mixotrophically on succinate or bicarbonate with Mn(II), in Mn-limited chemostats [51]. Mn(II)-oxidizing bacterium Pseudomonas putida GB-1 from a freshwater environment was also identified [52][53][54][55].

Thiosulfate-Oxidizing Capacity
The growth and SO 4 2− production of all strains in a thiosulfate oxidation medium, which contained an inorganic carbon source and sodium thiosulfate as an energy source, were examined. Halomonas spp. IOP_6, IOP_14, IOP_19, and IOP_31; Pseudomonas spp. IOP_13 and IOP_25; Marinobacter spp. IOP_29 and IOP_41 in γ-proteobacteria and Thalassospira sp. IOP_1; Citromirobium sp. IOP_28; and Aurantimonas sp. IOP_38 in αproteobacteria showed an autotrophic sulfur-oxidizing capacity with monomeric sulfur and/or sulfate produced.
The growth curves and the concentrations of electron donor S 2 O 3 2− and product SO 4 2− of strains Pseudomonas sp. IOP_13 and Halomonas sp. IOP_14 were further measured, as shown in Figure 4A,B. The results showed that the cell concentrations increased from the initial 10 5 cells/mL to the final 10 7 cells/mL after 8 days. The electron donor S 2 O 3 2− was consistently consumed by strains IOP_13 and IOP_14 after inoculation and produced SO 4 2− (287 mg/L, 394 mg/L) without SO 3 2− detected. The pH value of the culture increased during the first 3 days (0.2 units and 0.5 units) and then stabilized ( Figure 4C). This indicated that these strains produced alkaline substances in the process of sulfur oxidation. initial 10 5 cells/mL to the final 10 7 cells/mL after 8 days. The electron donor S2O3 2− was consistently consumed by strains IOP_13 and IOP_14 after inoculation and produced SO4 2− (287 mg/L, 394 mg/L) without SO3 2− detected. The pH value of the culture increased during the first 3 days (0.2 units and 0.5 units) and then stabilized ( Figure 4C). This indicated that these strains produced alkaline substances in the process of sulfur oxidation.

Carbon Dioxide-Fixation Capacity
All the strains were cultured in a sulfur-containing medium that used NaHCO 3 as the sole carbon source. The results showed that 12/17 strains exhibited autotrophic growth. Strain IOP_13 was chosen to culture with NaH 13 CO 3 as the sole carbon source to test its autotrophic capacity. The growth curve of strain IOP_13 and the value of δ13C in its biomass are shown in Figure 5. The results showed that bacterial cells increased from 10 5 to 10 7 cells/mL and that the δ13C VPDB value of the bacterium increased from −27.42‰ to 3460.06‰ within 11 days, which indicated that strain IOP_13 utilized inorganic carbon as a carbon source.

Genome Characteristics of Isolated Strains
The 17 isolates were sequenced with 300 × coverage, and the draft genomes were produced with 98.69%-100.00% completeness (Supplementary Table S2). The genome similarities between isolates and closest relatives were also analyzed, with 76.50%-100.00% ANI values and 65.88%-99.95% AAI values (Supplementary Table S1).
An analysis of the metabolic reconstruction using the KEGG database suggested that most of them have complete glycolysis, Entner-Doudoroff, pentosephosphate, and tricarboxylic acid cycle pathways, which showed their heterotrophic traits. The results for the functional gene annotation of strains are shown in Figure 6. Five strains, including Halomonas spp. IOP_6, IOP_14, and IOP_19 and Pseudomonas spp. IOP_13 and IOP_25, had high similarity with the sulfur-oxidation gene TsdA (Supplementary Figure S5). Tetrathionate-forming thiosulfate dehydrogenase (TsdA), identified by [56] from the purple sulfur bacterium Allochromatium vinosum, and this protein play the role of sulfur-based energy metabolism through the oxidation of thiosulfate. These isolates with the TsdA gene also showed a sulfur-oxidation ability, which could be carried out by the tetrasulphate pathway. Aurantimonas sp. IOP_38 and Seohaeicola sp. IOP_23 were annotated with the complete Sox sulfur-oxidation gene (SoxABCDHSFRSWXYZ). Strain IOP_38 could grow in an autotrophic sulfur-containing medium, but no sulfate or monosulfate production was detected. Seohaeicola sp. IOP_23 could grow only in a hetertrophic sulfur-containing medium.

Carbon Dioxide-Fixation Capacity
All the strains were cultured in a sulfur-containing medium that used NaHCO3 as the sole carbon source. The results showed that 12/17 strains exhibited autotrophic growth. Strain IOP_13 was chosen to culture with NaH 13 CO3 as the sole carbon source to test its autotrophic capacity. The growth curve of strain IOP_13 and the value of δ13C in its biomass are shown in Figure 5. The results showed that bacterial cells increased from 10 5 to 10 7 cells/mL and that the δ13CVPDB value of the bacterium increased from −27.42‰ to 3460.06‰ within 11 days, which indicated that strain IOP_13 utilized inorganic carbon as a carbon source.

Genome Characteristics of Isolated Strains
The 17 isolates were sequenced with 300 x coverage, and the draft genomes were produced with 98.69%-100.00% completeness (Supplementary Table S2). The genome similarities between isolates and closest relatives were also analyzed, with 76.50%-100.00% ANI values and 65.88%-99.95% AAI values (Supplementary Table S1).
An analysis of the metabolic reconstruction using the KEGG database suggested that most of them have complete glycolysis, Entner-Doudoroff, pentosephosphate, and tricarboxylic acid cycle pathways, which showed their heterotrophic traits. The results for the functional gene annotation of strains are shown in Figure 6. Five strains, including Halomonas spp. IOP_6, IOP_14, and IOP_19 and Pseudomonas spp. IOP_13 and IOP_25, The genes-encoding multicopper oxidases CumA [57] (Supplementary Figure S6) with the possible involvement in Mn(II) oxidation were found in strains IOP_13, IOP_16 and IOP_25, which have the capacity for Mn(II) oxidation.

Widespread of Mixotrophic Bacterial Strains in the Hydrothermal Vents
There is rich heterotrophic microbial life in hydrothermal vents. Versatile heterotrophic α-and γ-proteobacteria have been found in different venting areas of the Menez Gwen hydrothermal field in the Mid-Atlantic Ridge from the diffuse fluid discharge points through the mixing gradients to the plumes and the surrounding seawater [70]. Generalist species belonging to the genera Marinobacter, Vibrio, Pseudoalteromonas, Halomonas, Pseudomonas, and Alcanivorax, among others, have been repeatedly isolated from hydrothermal vent samples in the Pacific [4,[71][72][73][74], and their abundance in vent fluids collected from the Pacific Ocean was estimated to be up to 28 % of the total micro-organisms [75]. Phylotypes closely related to cultured species, e.g., Alteromonas, Halomonas, and Marinobacter, were relatively abundant in some crustal fluid samples in the Suiyo seamount off the eastern coast of Japan [76]. In this study, those heterotrophic α-and γ-proteobacteria were isolated from mussels, sulfides, hydrothermal sediments, and hydrothermal plumes around active hydrothermal vents (Table 1), which also indicated that they are widespread in these types of environments.

Widespread of Mixotrophic Bacterial Strains in the Hydrothermal Vents
There is rich heterotrophic microbial life in hydrothermal vents. Versatile heterotrophic αand γ-proteobacteria have been found in different venting areas of the Menez Gwen hydrothermal field in the Mid-Atlantic Ridge from the diffuse fluid discharge points through the mixing gradients to the plumes and the surrounding seawater [70]. Generalist species belonging to the genera Marinobacter, Vibrio, Pseudoalteromonas, Halomonas, Pseudomonas, and Alcanivorax, among others, have been repeatedly isolated from hydrothermal vent samples in the Pacific [4,[71][72][73][74], and their abundance in vent fluids collected from the Pacific Ocean was estimated to be up to 28% of the total micro-organisms [75]. Phylotypes closely related to cultured species, e.g., Alteromonas, Halomonas, and Marinobacter, were relatively abundant in some crustal fluid samples in the Suiyo seamount off the eastern coast of Japan [76]. In this study, those heterotrophic αand γ-proteobacteria were isolated from mussels, sulfides, hydrothermal sediments, and hydrothermal plumes around active hydrothermal vents (Table 1), which also indicated that they are widespread in these types of environments.
Compared with other Proteobacterial classes, ζ-proteobacterial populations as iron oxidizers have a narrow growth range and spread only in oxic-anoxic transition zones near shore environments [77] and iron-rich hydrothermal systems, such as iron-oxide material, hydrothermal sediment, etc. [78,79]. Until now, only seven species in two genera, Mariprofundus [80] and Ghiorsea [81], had been isolated, which have a narrow growth range of oxygen concentration in 0.07-2.0 µM [77]. Heterotrophic αand γ-proteobacteria shown across broad environmental gradients and dominate in the hydrothermal plumes, sulfides, and sediments might have important roles in the element cycle of iron and sulfur, which have been under estimation before.

Diverse Metabolism of Mixotrophic Bacterial Strains in the Hydrothermal Vents
Theoretical studies demonstrate that mixotrophy is advantageous in oligotrophic or fluctuating environments [82]. Micro-organisms survive using an adaptation ability for effective competition in the hydrothermal ecosystem with shifting biogeochemical conditions [83,84]. For example, archaea Pyrobaculum islandicum from hydrothermal vents can live as heterotrophs or autotrophs strictly under piezophilic conditions. The SUP05 clade of gammaproteobacteria (Thioglobaceae) could act as abundant autotrophs in the hydrothermal fluids and in association with eukaryotes at hydrothermal vents or as heterotrophes throughout the ocean [85]. The heterotrophic genera Pseudomonas, Halomonas, and Bacillus were all reported to possess the tetrathionate-forming ability using thiosulfate as a supplemental inorganic energy source [86,87]. Many strains in the Pseudomonas genus, such as strains of Pseudomonas stutzeri, were shown to oxidize sodium sulfide, change thiosulfate into tetrathionate, or grow on a variety of substrates, such as FeCl 2, FeCO 3 and FeSO 4 , as their sole energy source under anaerobically in the presence of nitrate [88][89][90]. We further predicted the sulfur-oxidizing and denitrification genes in 317 genomes belonging Pseudomonas in UniProt (http://sparql.uniprot.org/ accessed on 10 November 2022). It showed that 10 species, including P. stutzeri strain A1501, P. aeruginosa, P. veronii, P. wadenswilerensis, P. reidholzensis, P. fluorescens, P. extremaustralis, P. marincola, Pseudomonas sp. IsoF, and Pseudomonas sp. 9Ag-encoded TsdAB genes and NarI, were detected in 115 Pseudomonas genomes. It indicated that some Pseudomonas spp. have thiosulfate-oxidizing and nitratereducing potential for mixotrophic life. Some micro-organisms have been characterized as heterotrophes, but their autotrophic ability was not shown before. This study revealed their versatile metabolism as autotrophes and heterotrophes with sulfur and iron oxidization in aerobic-microaerobic-anaerobic conditions, and it further predicted possible mechanisms of these processes based on the annotation of their genomes. Sulfur oxidation with the tetrasulphate pathway by thiosulfate dehydrogenase was found common in Halomonas and Pseudomonas spp. of γ-proteobacteria, in this study. The thiosulfate-oxidizing enzyme (Sox) was found only in two isolates (Seohaeicola sp. IOP_23 and Aurantimonas sp. IOP_38) of α-proteobacteria. Almost all of them had genes for a complete pathway of denitrification. A total of 16 strains had c-type cytochromes maturation systems and c-type cytochromes with one or more heme-binding motifs, which may have a function in Fe redox reactions, as reported for iron oxidizers (Rhodobacter and Sideroxydans spp.) [62,64]. It is also noteworthy that Agromyces sp. IOP_2 in the phylum Actinobacteria with iron-and sulfur-oxidizing abilities lack all known iron-and sulfur-oxidizing genes, which needs to be further investigated.
However, it remains unknown how mixotrophic micro-organisms respond to different conditions across broad environmental gradients. The characterization of mixtrophs and their metabolisms deserve further attention.

Conclusions
With a rich variety of chemical energy sources and steep physical and chemical gradients, hydrothermal vent systems offer a range of habitats to support microbial life.
In this study, we isolated iron-oxidizing and thiosulfate-oxidizing bacteria from deepsea hydrothermal vents and shed light on the potential diverse functions of these heterotrophic bacteria in γand α-proteobacteria and Actinobacteria. Based on their broad growth ranges and versatile metabolisms, we predicted that heterotrophic bacteria which have capacities of sulfur, iron, and manganese oxidation may play important roles in carbon, sulfur, and metal cycling in hydrothermal vents.