Evidence for autotrophic growth of purple sulfur bacteria using pyrite as electron and sulfur source.

Purple sulfur bacteria (PSB) are capable of anoxygenic photosynthesis via oxidizing reduced sulfur compounds and are considered key drivers of the sulfur cycle in a range of anoxic environments. In this study, we show that Allochromatium vinosum (a PSB species) is capable of autotrophic growth using pyrite as the electron and sulfur source. Comparative growth profile, substrate characterization, and transcriptomic sequencing data provided valuable insight into the molecular mechanisms underlying the bacterial utilization of pyrite and autotrophic growth. Specifically, the pyrite-supported cell cultures ("py"') demonstrated robust but much slower growth rates and distinct patterns from their sodium sulfide-amended positive controls. Up to ~200-fold upregulation of genes encoding various c- and b-type cytochromes was observed in "py," pointing to the high relevance of these molecules in scavenging and relaying electrons from pyrite to cytoplasmic metabolisms. Conversely, extensive downregulation of genes related to LH and RC complex components indicates that the electron source may have direct control over the bacterial cells' photosynthetic activity. In terms of sulfur metabolism, genes encoding periplasmic or membrane-bound proteins (e.g., FccAB and SoxYZ) were largely upregulated, whereas those encoding cytoplasmic proteins (e.g., Dsr and Apr groups) are extensively suppressed. Other notable differentially expressed genes are related to flagella/fimbriae/pilin(+), metal efflux(+), ferrienterochelin(-), and [NiFe] hydrogenases(+). Characterization of the biologically reacted pyrite indicates the presence of polymeric sulfur. These results have, for the first time, put the interplay of PSB and transition metal sulfide chemistry under the spotlight, with the potential to advance multiple fields, including metal and sulfur biogeochemistry, bacterial extracellular electron transfer, and artificial photosynthesis. IMPORTANCE: Microbial utilization of solid-phase substrates constitutes a critical area of focus in environmental microbiology, offering valuable insights into microbial metabolic processes and adaptability. Recent advancements in this field have profoundly deepened our knowledge of microbial physiology pertinent to these scenarios and spurred innovations in biosynthesis and energy production. Furthermore, research into interactions between microbes and solid-phase substrates has directly linked microbial activities to the surrounding mineralogical environments, thereby enhancing our understanding of the relevant biogeochemical cycles. Our study represents a significant step forward in this field by demonstrating, for the first time, the autotrophic growth of purple sulfur bacteria using insoluble pyrite (FeS2) as both the electron and sulfur source. The presented comparative growth profiles, substrate characterizations, and transcriptomic sequencing data shed light on the relationships between electron donor types, photosynthetic reaction center activities, and potential extracellular electron transfer in these organisms capable of anoxygenic photosynthesis. Furthermore, the findings of our study may provide new insights into early-Earth biogeochemical evolutions, offering valuable constraints for understanding the environmental conditions and microbial processes that shaped our planet's history.

Complete genome of the thermophilic purple sulfur Bacterium Thermochromatium tepidum compared to Allochromatium vinosum and other Chromatiaceae.

The complete genome sequence of the thermophilic purple sulfur bacterium Thermochromatium tepidum strain MCT (DSM 3771T) is described and contrasted with that of its mesophilic relative Allochromatium vinosum strain D (DSM 180T) and other Chromatiaceae. The Tch. tepidum genome is a single circular chromosome of 2,958,290 base pairs with no plasmids and is substantially smaller than the genome of Alc. vinosum. The Tch. tepidum genome encodes two forms of RuBisCO and contains nifHDK and several other genes encoding a molybdenum nitrogenase but lacks a gene encoding a protein that assembles the Fe-S cluster required to form a functional nitrogenase molybdenum-iron cofactor, leaving the phototroph phenotypically Nif-. Tch. tepidum contains genes necessary for oxidizing sulfide to sulfate as photosynthetic electron donor but is genetically unequipped to either oxidize thiosulfate as an electron donor or carry out assimilative sulfate reduction, both of which are physiological hallmarks of Alc. vinosum. Also unlike Alc. vinosum, Tch. tepidum is obligately phototrophic and unable to grow chemotrophically in darkness by respiration. Several genes present in the Alc. vinosum genome that are absent from the genome of Tch. tepidum likely contribute to the major physiological differences observed between these related purple sulfur bacteria that inhabit distinct ecological niches.

Allochromatium tepidum, sp. nov., a hot spring species of purple sulfur bacteria.

We describe a new species of purple sulfur bacteria (Chromatiaceae, anoxygenic phototrophic bacteria) isolated from a microbial mat in the sulfidic geothermal outflow of a hot spring in Rotorua, New Zealand. This phototroph, designated as strain NZ, grew optimally near 45 °C but did not show an absorption maximum at 915 nm for the light-harvesting-reaction center core complex (LH1-RC) characteristic of other thermophilic purple sulfur bacteria. Strain NZ had a similar carotenoid composition as Thermochromatium tepidum, but unlike Tch. tepidum, grew photoheterotrophically on acetate in the absence of sulfide and metabolized thiosulfate. The genome of strain NZ was significantly larger than that of Tch. tepidum but slightly smaller than that of Allochromatium vinosum. Strain NZ was phylogenetically more closely related to mesophilic purple sulfur bacteria of the genus Allochromatium than to Tch. tepidum. This conclusion was reached from phylogenetic analyses of strain NZ genes encoding 16S rRNA and the photosynthetic functional gene pufM, from phylogenetic analyses of entire genomes, and from a phylogenetic tree constructed from the concatenated sequence of 1090 orthologous proteins. Moreover, average nucleotide identities and digital DNA:DNA hybridizations of the strain NZ genome against those of related species of Chromatiaceae supported the phylogenetic analyses. From this collection of properties, we describe strain NZ here as the first thermophilic species of the genus Allochromatium, Allochromatium tepidum NZT, sp. nov.

Phylogenomic analysis of a metagenome-assembled genome indicates a new taxon of an anoxygenic phototroph bacterium in the family Chromatiaceae and the proposal of "Candidatus Thioaporhodococcus" gen. nov.

In this study, three metagenome-assembled genomes of a sediment sample were constructed. A Bin1 (JB001) genome was identified as a photo-litho-auto/heterotroph (purple sulfur bacteria) bacterium with the ability to fix nitrogen, tolerate salt, and to produce bacteriochlorophyll a. It has a genome length of 4.1 Mb and a G + C content of 64.9%. Phylogenetic studies based on concatenated 92 core genes and photosynthetic genes (pufLM and bchY) showed that Bin JB001 is related to Thiococcus pfennigii, "Thioflavicoccus mobilis" and to the Lamprocystis purpurea lineage. Bin JB001 and its closely related members were subjected to the genome-based study of phenotypic and phylogenomic analysis. Genomic similarity indices (dDDH and ANI) showed that Bin JB001 could be defined as a novel species. The average amino acid identity (AAI) and percentage of conserved proteins (POCP) values were below 60 and 50%, respectively. The pan-genome analysis indicated that the pan-genome was an open type wherein Bin JB001 had 855 core genes. This study shows that the binned genome, Bin JB001 could represent a novel species of a new genus under the family Chromatiaceae, for which the name "Candidatus Thioaporhodococcus sediminis" gen. nov. sp. nov. is proposed.

Selective Excitation of Carotenoids of the Allochromatium vinosum Light-Harvesting LH2 Complexes Leads to Oxidation of Bacteriochlorophyll.

The mechanism of bacteriochlorophyll photooxidation in light-harvesting complexes of a number of purple photosynthetic bacteria when the complexes are excited into the carotenoid absorption bands remains unclear for many years. Here, using narrow-band laser illumination we measured action spectrum of this process for the spectral ranges of carotenoid and bacteriochlorophyll. It is shown that bacteriochlorophyll excitation results in almost no photooxidation of these molecules, while carotenoid excitation leads to oxidation with quantum yield of about 0,0003. Low value of the yield enabled an assumption that the studied process is initiated by the triplet states of the main carotenoids of the complexes with the number of conjugated double-bond chain length of N = 11. Interaction of these states with oxygen facilitates formation, though with low efficiency, of the excited singlet oxygen, which oxidizes bacteriochlorophylls. The carotenoid triplet states are formed in the process of the earlier studied singlet-triplet fission. The obtained results point at the necessity of reconsidering the functions of carotenoids in the light-harvesting complexes of purple bacteria.

The genome sequence of the giant phototrophic gammaproteobacterium Thiospirillum jenense gives insight into its physiological properties and phylogenetic relationships.

In a conserved culture of the purple sulfur bacterium Thiospirillum jenense DSM216T, cells of this species were easily recognized by cell morphology, large-size spirilla and visible flagellar tuft. The Tsp. jenense genome is 3.22 Mb in size and has a GC content of 48.7 mol%. It was readily identified as a member of the Chromatiaceae by the complement of proteins in its genome. A whole genome comparison clearly placed Tsp. jenense near Thiorhodovibrio and Rhabdochromatium species and somewhat more distant from Thiohalocapsa and Halochromatium species. This relationship was also found with the sequences of the photosynthetic reaction center protein PufM. The genome sequence supported important properties of this bacterium: the presence of ribulose-bisphosphate carboxylase and enzymes of the Calvin cycle of autotrophic carbon dioxide fixation but the absence of carboxysomes, an incomplete tricarboxylic acid cycle and the lack of malate dehydrogenase, the presence of a sulfur oxidation pathway including adenylylsulfate reductase (aprAB) but absence of assimilatory sulfate reduction, the presence of hydrogenase (hoxHMFYUFE), nitrogenase and a photosynthetic gene cluster (pufBALMC). The FixNOP type of cytochrome oxidase was notably lacking, which may be the reason that renders the cells highly sensitive to oxygen. Two minor phototrophic contaminants were found using metagenomic binning: one was identified as a strain of Rhodopseudomonas palustris and the second one has an average nucleotide identity of 82% to the nearest neighbor Rhodoferax antarcticus. It should be considered as a new species of this genus and Rhodoferax jenense is proposed as the name.

Spatiotemporal Changes in the Bacterial Community of the Meromictic Lake Uchum, Siberia.

Lake Uchum is a newly defined meromictic lake in Siberia with clear seasonal changes in its mixolimnion. This study characterized the temporal dynamics and vertical profile of bacterial communities in oxic and anoxic zones of the lake across all four seasons: October (autumn), March (winter), May (spring), and August (summer). Bacterial richness and diversity in the anoxic zone varied widely between time points. Proteobacteria was the dominant bacterial phylum throughout the oxic and anoxic zones across all four seasons. Alphaproteobacteria (Loktanella) and Gammaproteobacteria (Aliidiomarina) exhibited the highest abundance in the oxic and anoxic zone, respectively. Furthermore, there was a successional shift in sulfate-reducing bacteria (SRB) and sulfur-oxidizing bacteria in the anoxic zone across the seasons. The most dominant SRB, Desulfonatronovibrio sp., is likely one of the main producers of hydrogen sulfide (H2S) and typically accumulates the most H2S in winter. The representative anoxygenic phototrophic bacterial group in Lake Uchum was purple sulfur bacteria (PSB). PSB were dominant (60.76%) in summer, but only had 0.2-1.5% relative abundance from autumn to spring. Multivariate analysis revealed that the abundance of these SRB and PSB correlated to the concentration of H2S in Lake Uchum. Taken together, this study provides insights into the relationships between changes in bacterial community and environmental features in Lake Uchum.

Properties and structure of a low-potential, penta-heme cytochrome c552 from a thermophilic purple sulfur photosynthetic bacterium Thermochromatium tepidum.

The thermophilic purple sulfur bacterium Thermochromatium tepidum possesses four main water-soluble redox proteins involved in the electron transfer behavior. Crystal structures have been reported for three of them: a high potential iron-sulfur protein, cytochrome c', and one of two low-potential cytochrome c552 (which is a flavocytochrome c) have been determined. In this study, we purified another low-potential cytochrome c552 (LPC), determined its N-terminal amino acid sequence and the whole gene sequence, characterized it with absorption and electron paramagnetic spectroscopy, and solved its high-resolution crystal structure. This novel cytochrome was found to contain five c-type hemes. The overall fold of LPC consists of two distinct domains, one is the five heme-containing domain and the other one is an Ig-like domain. This provides a representative example for the structures of multiheme cytochromes containing an odd number of hemes, although the structures of multiheme cytochromes with an even number of hemes are frequently seen in the PDB database. Comparison of the sequence and structure of LPC with other proteins in the databases revealed several characteristic features which may be important for its functioning. Based on the results obtained, we discuss the possible intracellular function of this LPC in Tch. tepidum.

New proteins involved in sulfur trafficking in the cytoplasm of Allochromatium vinosum.

The formation of periplasmic sulfur globules is an intermediate step during the oxidation of reduced sulfur compounds in various sulfur-oxidizing microorganisms. The mechanism of how this sulfur is activated and crosses the cytoplasmic membrane for further oxidation to sulfite by the dissimilatory reductase DsrAB is incompletely understood, but it has been well documented that the pathway involves sulfur trafficking mediated by sulfur-carrying proteins. So far sulfur transfer from DsrEFH to DsrC has been established. Persulfurated DsrC very probably serves as a direct substrate for DsrAB. Here, we introduce further important players in oxidative sulfur metabolism; the proteins Rhd_2599, TusA, and DsrE2 are strictly conserved in the Chromatiaceae, Chlorobiaceae, and Acidithiobacillaceae families of sulfur-oxidizing bacteria and are linked to genes encoding complexes involved in sulfur oxidation (Dsr or Hdr) in the latter two. Here we show via relative quantitative real-time PCR and microarray analysis an increase of mRNA levels under sulfur-oxidizing conditions for rhd_2599, tusA, and dsrE2 in Allochromatium vinosum. Transcriptomic patterns for the three genes match those of major genes for the sulfur-oxidizing machinery rather than those involved in biosynthesis of sulfur-containing biomolecules. TusA appears to be one of the major proteins in A. vinosum. A rhd_2599-tusA-dsrE2-deficient mutant strain, although not viable in liquid culture, was clearly sulfur oxidation negative upon growth on solid media containing sulfide. Rhd_2599, TusA, and DsrE2 bind sulfur atoms via conserved cysteine residues, and experimental evidence is provided for the transfer of sulfur between these proteins as well as to DsrEFH and DsrC.

Carotenoid-dependent singlet oxygen photogeneration in light-harvesting complex 2 of Ectothiorhodospira haloalkaliphila leads to the formation of organic hydroperoxides and damage to both pigments and protein matrix.

Earlier, it was suggested that carotenoids in light-harvesting complexes 2 (LH2) can generate singlet oxygen, further oxidizing bacteriochlorophyll to 3-acetyl-chlorophyll. In the present work, it was found that illumination of isolated LH2 preparations of purple sulfur bacterium Ectothiorhodospira haloalkaliphila with light in the carotenoid absorption region leads to the photoconsumption of molecular oxygen, which is accompanied by the formation of hydroperoxides of organic molecules in the complexes. Photoformation of two types of organic hydroperoxides were revealed: highly lipophilic (12 molecules per one LH2) and relatively hydrophobic (68 per one LH2). It has been shown that illumination leads to damage to light-harvesting complexes. On the one hand, photobleaching of bacteriochlorophyll and a decrease in its fluorescence intensity are observed. On the other hand, the photoinduced increase in the hydrodynamic radius of the complexes, the reduction in their thermal stability, and the change in fluorescence intensity indicate conformational changes occurring in the protein molecules of the LH2 preparations. Inhibition of the processes described above upon the addition of singlet oxygen quenchers (L-histidine, Trolox, sodium L-ascorbate) may support the hypothesis that carotenoids in LH2 preparations are capable of generating singlet oxygen, which, in turn, damage to protein molecules.

Ectothiorhodospira lacustris sp. nov., a New Purple Sulfur Bacterium from Low-Mineralized Soda Lakes That Contains a Unique Pathway for Nitric Oxide Reduction.

Several strains of a Gram-negative, anaerobic photoautotrophic, motile, rod-shaped bacterium, designated as B14B, A-7R, and A-7Y were isolated from biofilms of low-mineralized soda lakes in central Mongolia and Russia (southeast Siberia). They had lamellar stacks as photosynthetic structures and bacteriochlorophyll a as the major photosynthetic pigment. The strains were found to grow at 25-35 °C, pH 7.5-10.2 (optimum, pH 9.0), and with 0-8% (w/v) NaCl (optimum, 0%). In the presence of sulfide and bicarbonate, acetate, butyrate, yeast extract, lactate, malate, pyruvate, succinate, and fumarate promoted growth. The DNA G + C content was 62.9-63.0 mol%. While the 16S rRNA gene sequences confirmed that the new strains belonged to the genus Ectothiorhodospira of the Ectothiorhodospiraceae, comparison of the genome nucleotide sequences of strains B14B, A-7R, and A-7Y revealed that the new isolates were remote from all described Ectothiorhodospira species both in dDDH (19.7-38.8%) and in ANI (75.0-89.4%). The new strains are also genetically differentiated by the presence of a nitric oxide reduction pathway that is lacking from all other Ectiothiorhodospiraceae. We propose to assign the isolates to the new species, Ectothiorhodospira lacustris sp. nov., with the type strain B14BT (=DSM 116064T = KCTC 25542T = UQM 41491T).

Fluorescence Microscopy Study of the Intracellular Sulfur Globule Protein SgpD in the Purple Sulfur Bacterium Allochromatium vinosum.

When oxidizing reduced sulfur compounds, the phototrophic sulfur bacterium Allochromatium vinosum forms spectacular sulfur globules as obligatory intracellular-but extracytoplasmic-intermediates. The globule envelope consists of three extremely hydrophobic proteins: SgpA and SgpB, which are very similar and can functionally replace each other, and SgpC which is involved in the expansion of the sulfur globules. The presence of a fourth protein, SgpD, was suggested by comparative transcriptomics and proteomics of purified sulfur globules. Here, we investigated the in vivo function of SgpD by coupling its carboxy-terminus to mCherry. This fluorescent protein requires oxygen for chromophore maturation, but we were able to use it in anaerobically growing A. vinosum provided the cells were exposed to oxygen for one hour prior to imaging. While mCherry lacking a signal peptide resulted in low fluorescence evenly distributed throughout the cell, fusion with SgpD carrying its original Sec-dependent signal peptide targeted mCherry to the periplasm and co-localized it exactly with the highly light-refractive sulfur deposits seen in sulfide-fed A. vinosum cells. Insertional inactivation of the sgpD gene showed that the protein is not essential for the formation and degradation of sulfur globules.

Thiorhodovibrio frisius and Trv. litoralis spp. nov., Two Novel Members from a Clade of Fastidious Purple Sulfur Bacteria That Exhibit Unique Red-Shifted Light-Harvesting Capabilities.

In the pursuit of cultivating anaerobic anoxygenic phototrophs with unusual absorbance spectra, a purple sulfur bacterium was isolated from the shoreline of Baltrum, a North Sea island of Germany. It was designated strain 970, due to a predominant light harvesting complex (LH) absorption maximum at 963-966 nm, which represents the furthest infrared-shift documented for such complexes containing bacteriochlorophyll a. A polyphasic approach to bacterial systematics was performed, comparing genomic, biochemical, and physiological properties. Strain 970 is related to Thiorhodovibrio winogradskyi DSM 6702T by 26.5, 81.9, and 98.0% similarity via dDDH, ANI, and 16S rRNA gene comparisons, respectively. The photosynthetic properties of strain 970 were unlike other Thiorhodovibrio spp., which contained typical LH absorbing characteristics of 800-870 nm, as well as a newly discovered absorption band at 908 nm. Strain 970 also had a different photosynthetic operon composition. Upon genomic comparisons with the original Thiorhodovibrio strains DSM 6702T and strain 06511, the latter was found to be divergent, with 25.3, 79.1, and 97.5% similarity via dDDH, ANI, and 16S rRNA gene homology to Trv. winogradskyi, respectively. Strain 06511 (=DSM 116345T) is thereby described as Thiorhodovibrio litoralis sp. nov., and the unique strain 970 (=DSM 111777T) as Thiorhodovibrio frisius sp. nov.

Genomic Comparison, Phylogeny and Taxonomic Reevaluation of the Ectothiorhodospiraceae and Description of Halorhodospiraceae fam. nov. and Halochlorospira gen. nov.

The Ectothiorhodospiraceae family represents purple sulfur bacteria of the Gammaproteobacteria found primarily in alkaline soda lakes of moderate to extremely high salinity. The main microscopically visible characteristic separating them from the Chromatiaceae is the excretion of the intermediate elemental sulfur formed during oxidation of sulfide prior to complete oxidation to sulfate rather than storing it in the periplasm. We present a comparative study of 38 genomes of all species of phototrophic Ectothiorhodospiraceae. We also include a comparison with those chemotrophic bacteria that have been assigned to the family previously and critically reevaluate this assignment. The data demonstrate the separation of Halorhodospira species in a major phylogenetic branch distant from other Ectothiorhodospiraceae and support their separation into a new family, for which the name Halorhodospiraceae fam. nov. is proposed. In addition, the green-colored, bacteriochlorophyll-containing species Halorhodospira halochloris and Halorhodospira abdelmalekii were transferred to the new genus Halochlorospira gen. nov. of this family. The data also enable classification of several so far unclassified isolates and support the separation of Ectothiorhodospira shaposhnikovii and Ect. vacuolata as well as Ect. mobilis and Ect. marismortui as distinct species.

Light attenuation in enriched purple phototrophic bacteria cultures: Implications for modelling and reactor design.

Light attenuation in enriched purple phototrophic bacteria (PPB) cultures has not been studied, and its understanding is critical for proper process modelling and reactor design, especially for scaled systems. This work evaluated the effect of different biomass concentrations, reactor configurations, wastewater matrices, and growth conditions, on the attenuation extent of near infra-red (NIR) and ultraviolet-visible (UV-VIS) light spectra. The results show that increased biomass concentrations lead to higher light attenuation, and that PPB absorb both VIS and NIR wavelengths, with both fractions of the spectrum being equally absorbed at biomass concentrations above 1,000 g COD·m-3. A flat plate configuration showed less attenuation compared with cylindrical reactors illuminated from the top, representative for open ponds. Neither a complex wastewater matrix nor the presence of polyhydroxyalkanoates (under nutrient limited conditions) affected light attenuation significantly. The pigment concentration (both bacteriochlorophyll and carotenoids) however, had a strong effect, with significant attenuation in the presence of pigments. Attenuation predictions using the Lambert-Beer law (excluding scattering) and the Schuster model (including scattering) indicated that light scattering had a minimal effect. A proposed mathematical model, based on the Lambert-Beer law and a Monod function for light requirements, allowed effective prediction of the kinetics of photoheterotrophic growth. This resulted in a half saturation coefficient of 4.6 W·m-2. Finally, the results showed that in dense outdoor PPB cultures (≥1,000 g COD·m-3), effective light penetration is only 5 cm, which biases design away from horizontal lagoons, and towards non-incident multi-panel systems such as flat plate reactors.

Single-cell genomics-based analysis reveals a vital ecological role of Thiocapsa sp. LSW in the meromictic Lake Shunet, Siberia.

Meromictic lakes usually harbour certain prevailing anoxygenic phototrophic bacteria in their anoxic zone, such as the purple sulfur bacterium (PSB) Thiocapsa sp. LSW (hereafter LSW) in Lake Shunet, Siberia. PSBs have been suggested to play a vital role in carbon, nitrogen and sulfur cycling at the oxic-anoxic interface of stratified lakes; however, the ecological significance of PSBs in the lake remains poorly understood. In this study, we explored the potential ecological role of LSW using a deep-sequencing analysis of single-cell genomics associated with flow cytometry. An approximately 2.7 Mb draft genome was obtained based on the co-assembly of five single-cell genomes. LSW might grow photolithoautotrophically and could play putative roles not only as a carbon fixer and diazotroph, but also as a sulfate reducer/oxidizer in the lake. This study provides insights into the potential ecological role of Thiocapsa sp. in meromictic lakes.

Microbial Community Composition during a Bloom of Purple Bacteria in Intertidal Sediments in Vigo (Northwest Spain).

In summer 2019, a large, bright pink microbial mat was visible on top of macroalgal deposits in muddy sediments of an urban beach (Playa do Adro, Vigo). In order to characterize the dominant organisms in these colored mats, results from microscopic observations, photosynthetic pigments, and molecular analysis were gathered. Light microscopy examination revealed pinkish microbial aggregates with minor contributions of larger protists and cyanobacteria. High-performance liquid chromatography (HPLC) pigment analysis documented the dominance of bacteriochlorophyll a and carotenoids whose spectra were compatible with those described in photosynthetic purple bacteria. 16S rRNA gene amplicon sequencing confirmed that the vast majority of reads belonged to Proteobacteria (73.5%), and among them, nearly 88% of those reads belonged to purple sulfur bacteria (Gammaproteobacteria). A single family, Chromatiaceae, constituted the bulk of this assemblage, including the genera Thiohalocapsa (32%), Marichromatium (12.5%), Phaeochromatium (5%), and Halocromatium (2%) as main contributors. Nonetheless, a considerable number of sequences could not be assigned to a particular genus, stressing the large biological diversity in these microbial mats and the potential presence of novel taxa of purple sulfur bacteria. IMPORTANCE Urban beaches are valuable recreational areas particularly vulnerable to human disturbance. In these areas, the intertidal sediments harbor a diverse community of microorganisms, including virus, bacteria, fungi, and protozoa. In this sense, pollution events can introduce pathogenic allochthonous microbes which may constitute a human health risk. Visual and sensory observations, such as a weird color or bad smell, are usually appreciated as a warning by beachgoers and authorities, as indeed was the case at do Adro beach in 2019. The observed proliferation seems to be common in summertime, but its dimension alerted beachgoers and media. The obtained results allowed for the identification of purple sulfur bacteria as responsible for the pink-violet top layer staining the intertidal zone. These blooms have never been associated with public health risks. Beyond solving the sanitary concern, other important findings were its diversity and large proportion of novel taxa, illustrating the complexity of these ecosystems.

The HydS C-terminal domain of the Thiocapsa bogorovii HydSL hydrogenase is involved in membrane anchoring and electron transfer.

Thiocapsa bogorovii BBS (former name Thiocapsa roseopersicina) contains HydSL hydrogenase belonging to 1e subgroup of NiFe hydrogenases (isp-type). The operon of these hydrogenases contains gene for small subunit (hydS), gene for large subunit (hupL), and genes isp1 and isp2 between them. It is predicted that last two genes code electron transport careers for electron transfer from/to HydSL hydrogenase. However, the interaction between them is unclear. The aim of this study was to determine structural and functional role of T. bogorovii HydS C-terminal end. For this purpose, we modelled all subunits of the complex HydS-HydL-Isp1-Isp2. Hydrophobicity surface analysis of the Isp1 model revealed highly hydrophobic helices suggesting potential membrane localization, as well as the hydrophilic C-terminus, which is likely localized outside of membrane. Isp1 model was docked with models of full length and C-terminal truncated HydSL hydrogenases and results illustrate the possibility of HydSL membrane anchoring via transmembrane Isp1 with essential participation of C-terminal end of HydS in the interaction. C-terminal end of HydS subunit was deleted and our studies revealed that the truncated HydSL hydrogenase detached from cellular membranes in contrast to native hydrogenase. It is known that HydSL hydrogenase in T. bogorovii performs the reaction of elemental sulfur reduction (S0 + H2 = ≥H2S). Cells with truncated HydS produced much less H2S in the presence of H2 and S0. Thus, our data support the conclusion that C-terminal end of HydS subunit participates in interaction of HydSL hydrogenase with Isp1 protein for membrane anchoring and electron transfer.

Purple phototrophic bacteria are outcompeted by aerobic heterotrophs in the presence of oxygen.

There is an ongoing debate around the effect of microaerobic/aerobic conditions on the wastewater treatment performance and stability of enriched purple phototrophic bacteria (PPB) cultures. It is well known that oxygen-induced oxidative conditions inhibit the synthesis of light harvesting complexes, required for photoheterotrophy. However, in applied research, several publications have reported efficient wastewater treatment at high dissolved oxygen (DO) levels. This study evaluated the impact of different DO concentrations (0-0.25 mg·L-1, 0-0.5 mg·L-1 and 0-4.5 mg·L-1) on the COD, nitrogen and phosphorus removal performances, the biomass yields, and the final microbial communities of PPB-enriched cultures, treating real wastewaters (domestic and poultry processing wastewater). The results show that the presence of oxygen suppressed photoheterotrophic growth, which led to a complete pigment and colour loss in a matter of 20-30 h after starting the batch. Under aerobic conditions, chemoheterotrophy was the dominant catabolic pathway, with wastewater treatment performances similar to those achieved in common aerobic reactors, rather than those corresponding to phototrophic systems (i.e. considerable total COD decrease (45-57% aerobically vs. ± 10% anaerobically). This includes faster consumption of COD and nutrients, lower nutrient removal efficiencies (50-58% vs. 72-99% for NH4+-N), lower COD:N:P substrate ratios (100:4.5-5.0:0.4-0.8 vs. 100:6.7-12:0.9-1.2), and lower apparent biomass yields (0.15-0.31 vs. 0.8-1.2 g CODbiomass·g CODremoved-1)). The suppression of photoheterotrophy inevitably resulted in a reduction of the relative PPB abundances in all the aerated tests (below 20% at the end of the tests), as PPB lost their main competitive advantage against competing aerobic heterotrophic microbes. This was explained by the lower aerobic PPB growth rates (2.4 d-1 at 35 °C) when compared to common growth rates for aerobic heterotrophs (6.0 d-1 at 20 °C). Therefore, PPB effectively outcompete other microbes under illuminated-anaerobic conditions, but not under aerobic or even micro-aerobic conditions, as shown by continuously aerated tests controlled at undetectable DO levels. While their aerobic heterotrophic capabilities provide some resilience, at non-sterile conditions PPB cannot dominate when growing chemoheterotrophically, and will be outcompeted.

Purple phototrophic bacteria for resource recovery: Challenges and opportunities.

Sustainable development is driving a rapid focus shift in the wastewater and organic waste treatment sectors, from a "removal and disposal" approach towards the recovery and reuse of water, energy and materials (e.g. carbon or nutrients). Purple phototrophic bacteria (PPB) are receiving increasing attention due to their capability of growing photoheterotrophically under anaerobic conditions. Using light as energy source, PPB can simultaneously assimilate carbon and nutrients at high efficiencies (with biomass yields close to unity (1 g CODbiomass·g CODremoved-1)), facilitating the maximum recovery of these resources as different value-added products. The effective use of infrared light enables selective PPB enrichment in non-sterile conditions, without competition with other phototrophs such as microalgae if ultraviolet-visible wavelengths are filtered. This review reunites results systematically gathered from over 177 scientific articles, aiming at producing generalized conclusions. The most critical aspects of PPB-based production and valorisation processes are addressed, including: (i) the identification of the main challenges and potentials of different growth strategies, (ii) a critical analysis of the production of value-added compounds, (iii) a comparison of the different value-added products, (iv) insights into the general challenges and opportunities and (v) recommendations for future research and development towards practical implementation. To date, most of the work has not been executed under real-life conditions, relevant for full-scale application. With the savings in wastewater discharge due to removal of organics, nitrogen and phosphorus as an important economic driver, priorities must go to using PPB-enriched cultures and real waste matrices. The costs associated with artificial illumination, followed by centrifugal harvesting/dewatering and drying, are estimated to be 1.9, 0.3-2.2 and 0.1-0.3 $·kgdry biomass-1. At present, these costs are likely to exceed revenues. Future research efforts must be carried out outdoors, using sunlight as energy source. The growth of bulk biomass on relatively clean wastewater streams (e.g. from food processing) and its utilization as a protein-rich feed (e.g. to replace fishmeal, 1.5-2.0 $·kg-1) appears as a promising valorisation route.