Hi! My name is Carolina Reyes and I am a microbiologist. I am interested in understanding the role of microorganisms in biogeochemical cycles including iron and copper cycles. Previous research focused on understanding the types of microorganisms involved in Fe and N-cycling in marine sediments and bacterial Fe-reduction and As-mobilization. Supervisors: Chad W. Saltikov, Michael W. Friedrich, Stephan M. Kraemer, Tina Salmassi, Jennifer Jay, and Christa Schleper
Cocultivation of fungi and algae can result in a mutualistic or antagonistic interaction dependin... more Cocultivation of fungi and algae can result in a mutualistic or antagonistic interaction depending on the species involved and the cultivation conditions. In this study, we investigated the growth behavior and enzymatic activity of two filamentous white-rot fungi (Trametes versicolor and Trametes pubescens) and two freshwater algae (Chlorella vulgaris and Scenedesmus vacuolatus) cocultured in the presence of TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl radical) oxidized cellulose nanofibrils (CNF) and cellulose nanocrystals (CNC). The growth of fungi and algae was studied in liquid, agar medium, and 3D-printed nanocellulose hydrogels. The results showed that cocultures grew faster under nutrient-rich conditions than in nutrient-depleted conditions. Key cellulose-degrading enzymes, including endoglucanase and laccase activities, were higher in liquid cocultures of T. versicolor and S. vacuolatus in the presence of cellulose compared to single cultures of fungi or algae. Although similar results were observed for cocultures of T. pubescens and C. vulgaris, laccase production diminished over time in these cultures. Fungi and algae were capable of growth in 3D-printed cellulose hydrogels. These results showed that cellulase enzyme production could be enhanced by cocultivating white-rot fungi with freshwater algae under nutrient-rich conditions with TEMPO-CNF and CNC. Additionally, the growth of white-rot fungi and freshwater algae in printed cellulose hydrogels demonstrates the potential use of fungi and algae in hydrogel systems for biotechnological applications, including biofuel production and bio-based fuel cell components.
White-rot fungi can degrade all lignocellulose components due to their potent lignin and cellulos... more White-rot fungi can degrade all lignocellulose components due to their potent lignin and cellulose-degrading enzymes. In this study, five white-rot fungi, Trametes versicolor, Trametes pubescens, Ganoderma adspersum, Ganoderma lipsiense, and Rigidoporus vitreus were tested for endoglucanase, laccase, urease, and glucose-6-phosphate (G6P) production when grown with malt extract and nanocellulose in the form of TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl radical) oxidized cellulose nanofibrils (CNF) and cellulose nanocrystals (CNC). Results show that temperature plays a key role in controlling the growth of all five fungi when cultured with malt extract alone. Endoglucanase activities were highest in cultures of G. adspersum and G. lipsiense and laccase activities were highest in cultures of T. versicolor and R. vitreus. Urease activities were highest in cultures of G. adspersum, G. lipsiense, and R. vitreus. Glucose-6-phosphate levels also indicate that cells were actively metabolizing glucose present in the cultures. These results show that TEMPO-oxidized CNF and CNC do not inhibit the production of specific lignocellulose enzymes by these white-rot fungi. The apparent lack of enzymatic inhibition makes TEMPO-oxidized CNF and CNC excellent candidates for future biotechnological applications in combination with the white-rot fungi studied here.
World Journal of Microbiology and Biotechnology volume, 2020
Melanins are natural biopolymers that are known to contribute to different biological processes a... more Melanins are natural biopolymers that are known to contribute to different biological processes and to protect organisms from adverse environmental conditions. During the past decade, melanins have attracted increasing attention for their use in organic semiconductors and bioelectronics, drug delivery, photoprotection and environmental bioremediation. Although considerable advances in these fields have been achieved, real-world applications of melanins are still scarce, probably due to the limited and expensive source of natural melanin. Nevertheless, recent biotechnological advances have allowed for relatively large-scale production of microbial melanins, which could replace current commercial melanin. In this review, we first describe different melanin sources and highlight the advantages and disadvantages of each production method. Our focus is on the microbial synthesis of melanins, including the methodology and mechanism of melanin formation. Applications of microbial melanins are also discussed, and an outlook on how to push the field forward is discussed.
Ammonia-oxidizing archaea (AOA) are widespread in nature and are involved in nitrification, an es... more Ammonia-oxidizing archaea (AOA) are widespread in nature and are involved in nitrification, an essential process in the global nitrogen cycle. The enzymes for ammonia oxidation and electron transport rely heavily on copper (Cu), which can be limited in nature. In this study the model soil archaeon Nitrososphaera viennensis was investigated via transcriptomic analysis to gain insight regarding possible Cu uptake mechanisms and compensation strategies when Cu becomes limiting. Upon Cu limitation, N. viennensis exhibited impaired nitrite production and thus growth, which was paralleled by downregulation of ammonia oxidation, electron transport, carbon fixation, nucleotide, and lipid biosynthesis pathway genes. Under Cu-limitation, 1547 out of 3180 detected genes were differentially expressed, with 784 genes upregulated and 763 downregulated. The most highly upregulated genes encoded proteins with a possible role in Cu binding and uptake, such as the Cu chelator and transporter CopC/D, disulfide bond oxidoreductase D (dsbD), and multicopper oxidases. While this response differs from the marine strain Nitrosopumilus maritimus, conserved sequence motifs in some of the Cu-responsive genes suggest conserved transcriptional regulation in terrestrial AOA. This study provides possible gene regulation and energy conservation mechanisms linked to Cu bioavailability and presents the first model for Cu uptake by a soil AOA.
Ammonia oxidizing archaea (AOA) inhabiting soils have a central role in the global nitrogen cycle... more Ammonia oxidizing archaea (AOA) inhabiting soils have a central role in the global nitrogen cycle. Copper (Cu) is central to many enzymes in AOA including ammonia monooxygenase (AMO), the enzyme involved in the first step of ammonia oxidation. This study explored the physiological response of the AOA soil isolate, Nitrososphaera viennensis (EN76T) to Cu-limiting conditions in order to approach its limiting threshold under laboratory conditions. The chelator TETA (1,4,8,11-tetraazacyclotetradecane N, N',N'',N'''-tetraacetic acid hydrochloride hydrate) with selective affinity for Cu2+ was used to lower bioavailable Cu2+ in culture experiments as predicted by thermodynamic speciation calculations. Results show that N. viennensis is Cu-limited at concentrations ≤10^-15 mol/L free Cu2+ compared to standard conditions (10^-12 mol/L). This Cu2+ limiting threshold is similar to pure cultures of denitrifying bacteria and other AOA and AOB inhabiting soils, freshwaters and sewage (<10^-16 mol/L), and lower than pure cultures of the marine AOA Nitrosopumilus maritimus (<10^-12.7 mol/L), which also possesses a high amount of Cu-dependent enzymes.
International Journal of Systematic and Evolutionary Microbiology , 2019
Two mesophilic, neutrophilic and aerobic marine ammonia-oxidizing archaea, designated strains NF5... more Two mesophilic, neutrophilic and aerobic marine ammonia-oxidizing archaea, designated strains NF5T and D3CT, were isolated from coastal surface water of the Northern Adriatic Sea. Cells were straight small rods 0.20–0.25 µm wide and 0.49–2.00 µm long. Strain NF5T possessed archaella as cell appendages. Glycerol dibiphytanyl glycerol tetraethers with zero to four cyclopentane moieties (GDGT-0 to GDGT-4) and crenarchaeol were the major core lipids. Menaquinone MK6 : 0 was the major respiratory quinone. Both isolates gained energy by oxidizing ammonia (NH3) to nitrite (NO2 -) and used bicarbonate as a carbon source. Strain D3CT was able use urea as a source of ammonia for energy production and growth. Addition of hydrogen peroxide (H2O2) scavengers (catalase or α-keto acids) was required to sustain growth. Optimal growth occurred between 30 and 32 °C, pH 7.1 and 7.3 and between 34 and 37‰ salinity. The cellular metal abundance ranking of both strains was Fe>Zn>Cu>Mn>Co. The genomes of strains NF5T and D3CT have a DNA G+C content of 33.4 and 33.8 mol%, respectively. Phylogenetic analyses of 16S rRNA gene sequences revealed that both strains are affiliated with the class Nitrososphaeria , sharing ~85 % 16S rRNA gene sequence identity with Nitrososphaera viennensis EN76T. The two isolates are separated by phenotypic and genotypic characteristics and are assigned to distinct species within the genus Nitrosopumilus gen. nov. according to average nucleotide identity thresholds of their closed genomes. Isolates NF5T (=JCM 32270T =NCIMB 15114T) and D3CT (=JCM 32271T =DSM 106147T =NCIMB 15115T) are type strains of the species Nitrosopumilus adriaticus sp. nov. and Nitrosopumilus piranensis sp. nov., respectively.
One sentence summary: This study gives insight into the bacterial community composition related t... more One sentence summary: This study gives insight into the bacterial community composition related to iron-cycling in suboxic marine sediments based on 16S rRNA gene pyrosequencing. ABSTRACT To gain insight into the bacterial communities involved in iron-(Fe) cycling under marine conditions, we analysed sediments with Fe-contents (0.5–1.5 wt %) from the suboxic zone at a marine site in the Skagerrak (SK) and a brackish site in the Bothnian Bay (BB) using 16S rRNA gene pyrosequencing. Several bacterial families, including Desulfobulbaceae, Desulfuromonadaceae and Pelobacteraceae and genera, including Desulfobacter and Geobacter, known to reduce Fe were detected and showed highest abundance near the Fe(III)/Fe(II) redox boundary. Additional genera with microorganisms capable of coupling fermentation to Fe-reduction, including Clostridium and Bacillus, were observed. Also, the Fe-oxidizing families Mariprofundaceae and Gallionellaceae occurred at the SK and BB sites, respectively, supporting Fe-cycling. In contrast, the sulphate (SO 4 2−) reducing bacteria Desulfococcus and Desulfobacterium were more abundant at greater depths concurring with a decrease in Fe-reducing activity. The communities revealed by pyrosequencing, thus, match the redox stratification indicated by the geochemistry, with the known Fe-reducers coinciding with the zone of Fe-reduction. Not the intensely studied model organisms, such as Geobacter spp., but rather versatile microorganisms, including sulphate reducers and possibly unknown groups appear to be important for Fe-reduction in these marine suboxic sediments.
The fate and transport of arsenic is regulated, in part, by its strong affinity for iron (hydr)ox... more The fate and transport of arsenic is regulated, in part, by its strong affinity for iron (hydr)oxides. A transition from aerobic to anaerobic conditions resulting in concomitant reduction of both As(V) and iron (hydr)oxides can thus have a pronounced influence on As partitioning. However, it is presently unclear whether As desorption under anaerobic conditions results predominantly from a transformation from As(V) to As(III) or from mineralogical changes as a consequence of iron and manganese reduction. Here, we examine desorption of both As(III) and As(V) from ferrihydrite-, goethite-, and hematite-coated sand under hydrodynamic conditions. Furthermore, to resolve the relative role of Fe(III) and/or As(V) reduction in regulating dissolved As concentrations, we also examined As desorption from ferrihydrite-and goethite-coated sands presorbed with As(V) using wild type or mutants of Shewanella sp. ANA-3, capable of Fe(III)-and/or As(V)-reduction. We reveal substantial differences in As(III) and As(V) desorption from ferrihydrite, goethite, and hematite. Despite being adsorbed to a greater extent than As(V), As(III) is desorbed more rapidly and extensively from all oxides, suggesting weaker binding of As(III) than As(V). When As(V) and Fe(III) reduction are decoupled, As(V) reduction appears to be the dominant process controlling As release. Our results also suggest the importance of appreciating physical properties of specific Fe (hydr)oxides when predicting the potential for As desorption.
Extracellular respiration of solid-phase electron acceptors in some microorganisms requires a com... more Extracellular respiration of solid-phase electron acceptors in some microorganisms requires a complex chain of multiheme ctype cytochromes that span the inner and outer membranes. In Shewanella species, MtrA, an35-kDa periplasmic decaheme c-type cytochrome, is an essential component for extracellular respiration of iron(III). The exact mechanism of electron transport has not yet been resolved, but the arrangement of the polypeptide chain may have a strong influence on the capability of the MtrA cytochrome to transport electrons. The iron hemes of MtrA are bound to its polypeptide chain via proximal (CXXCH) and distal histidine residues. In this study, we show the effects of mutating histidine residues of MtrA to arginine on protein expression and extracellular respiration using Shewanella sp. strain ANA-3 as a model organism. Individual mutations to six out of nine proximal histidines in CXXCH of MtrA led to decreased protein expression. However, distal histidine mutations resulted in various degrees of protein expression. In addition, the effects of histidine mutations on extracellular respiration were tested using ferrihydrite and current production in microbial fuel cells. These results show that proximal histidine mutants were unable to reduce ferrihydrite. Mutations to the distal histidine residues resulted in various degrees of ferrihydrite reduction. These findings indicate that mutations to the proximal histidine residues affect MtrA expression, leading to loss of extracellular respiration ability. In contrast, mutations to the distal histidine residues are less detrimental to protein expression, and extracellular respiration can proceed.
Arsenate respiration and Fe(III) reduction are important processes that influence the fate and tr... more Arsenate respiration and Fe(III) reduction are important processes that influence the fate and transport of arsenic in the environment. The goal of this study was to investigate the impact of arsenate on Fe(III) reduction using arsenate and Fe(III) reduction deficient mutants of Shewanella sp. strain ANA-3. Ferrihydrite reduction in the absence of arsenate was similar for an arsenate reduction mutant (arrA and arsC deletion strain of ANA-3) compared with wild-type ANA-3. However, the presence of arsenate adsorbed onto fer-rihydrite impeded Fe(III) reduction for the arsenate reduction mutant but not in the wild-type. In an Fe(III) reduction mutant (mtrDEF, omcA, mtrCAB null mutant of ANA-3), arsenate was reduced similarly to wild-type ANA-3 indicating the Fe(III) reduction pathway is not required for ferrihydrite-associated arsenate reduction. Expression analysis of the mtr/omc gene cluster of ANA-3 showed that omcA and mtrCAB were expressed under soluble Fe(III), ferrihydrite and arsenate growth conditions and not in aerobically grown cells. Expression of arrA was greater with ferrihydrite pre-adsorbed with arsenate relative to ferrihydrite only. Lastly, arrA and mtrA were simultaneously induced in cells shifted to anaerobic conditions and exposed to soluble Fe(III) and arsenate. These observations suggest that, unlike Fe(III), arsen-ate can co-induce operons (arr and mtr) implicated in arsenic mobilization.
In many marine surface sediments, the reduction of manganese (Mn) and iron (Fe) oxides is obscure... more In many marine surface sediments, the reduction of manganese (Mn) and iron (Fe) oxides is obscured by sulfate reduction, which is regarded as the predominant anaerobic microbial respiration process. However, many dissimilatory sulfate and sulfur reducing microorganisms are known to utilize alternative electron acceptors such as metal oxides. In this study, we tested whether sulfate and sulfur reducing bacteria are linked to metal oxide reduction based on biogeochemical modeling of porewater concentration profiles of Mn2+ and Fe2+ in Bothnian Bay (BB) and Skagerrak (SK) sediments. Steady-state modeling of Fe2+ and Mn2+ porewater profiles revealed zones of net Fe (0–9 cm BB; ∼10 and 20 cm SK) and Mn (0–5 cm BB; 2–8 cm SK) species transformations. 16S rRNA pyrosequencing analysis of the in-situ community showed that Desulfobacteraceae, Desulfuromonadaceae and Desulfobulbaceae were present in the zone of Fe-reduction of both sediments. Rhodobacteraceae were also detected at high relative abundance in both sediments. BB sediments appeared to harbor a greater diversity of potential Fe-reducers compared to SK. Additionally, when the upper 10 cm of sediment from the SK was incubated with lepidocrocite and acetate, Desulfuromonas was the dominant bacteria. Real-time quantitative polymerase chain reaction (qPCR) results showed decreasing dsrA gene copy numbers with depth coincided with decreased Fe-reduction activity. Our results support the idea that sulfur and sulfate reducing bacteria contribute to Fe-reduction in the upper centimeters of both sediments.
In this study, we analysed metagenomes along with biogeochemical profiles from Skagerrak (SK) and... more In this study, we analysed metagenomes along with biogeochemical profiles from Skagerrak (SK) and Bothnian Bay (BB) sediments, to trace the prevailing nitrogen pathways. NO 3 − was present in the top 5 cm below the sediment-water interface at both sites. NH 4 + increased with depth below 5 cm where it overlapped with the NO 3 − zone. Steady-state modelling of NO 3 − and NH 4 + porewater profiles indicates zones of net nitrogen species transformations. Bacterial protease and hydratase genes appeared to make up the bulk of total ammonification genes. Genes involved in ammonia oxidation (amo, hao), denitrification (nir, nor), dis-similatory NO 3 − reduction to NH 4 + (nfr and otr) and in both of the latter two pathways (nar, nap) were also present. Results show ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) are similarly abundant in both sediments. Also, denitrification genes appeared more abundant than DNRA genes. 16S rRNA gene analysis showed that the relative abundance of the nitrifying group Nitrosopumilales and other groups involved in nitrification and denitrification (Nitrobacter, Nitrosomonas, Nitrospira, Nitrosococcus and Nitrosomonas) appeared less abundant in SK sediments compared to BB sediments. Beggiatoa and Thiothrix 16S rRNA genes were also present, suggesting chemolithoautotrophic NO 3 − reduction to NO 2 − or NH 4 + as a possible pathway. Our results show the metabolic potential for ammonification, nitrification, DNRA and denitrification activities in North Sea and Baltic Sea sediments.
Cocultivation of fungi and algae can result in a mutualistic or antagonistic interaction dependin... more Cocultivation of fungi and algae can result in a mutualistic or antagonistic interaction depending on the species involved and the cultivation conditions. In this study, we investigated the growth behavior and enzymatic activity of two filamentous white-rot fungi (Trametes versicolor and Trametes pubescens) and two freshwater algae (Chlorella vulgaris and Scenedesmus vacuolatus) cocultured in the presence of TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl radical) oxidized cellulose nanofibrils (CNF) and cellulose nanocrystals (CNC). The growth of fungi and algae was studied in liquid, agar medium, and 3D-printed nanocellulose hydrogels. The results showed that cocultures grew faster under nutrient-rich conditions than in nutrient-depleted conditions. Key cellulose-degrading enzymes, including endoglucanase and laccase activities, were higher in liquid cocultures of T. versicolor and S. vacuolatus in the presence of cellulose compared to single cultures of fungi or algae. Although similar results were observed for cocultures of T. pubescens and C. vulgaris, laccase production diminished over time in these cultures. Fungi and algae were capable of growth in 3D-printed cellulose hydrogels. These results showed that cellulase enzyme production could be enhanced by cocultivating white-rot fungi with freshwater algae under nutrient-rich conditions with TEMPO-CNF and CNC. Additionally, the growth of white-rot fungi and freshwater algae in printed cellulose hydrogels demonstrates the potential use of fungi and algae in hydrogel systems for biotechnological applications, including biofuel production and bio-based fuel cell components.
White-rot fungi can degrade all lignocellulose components due to their potent lignin and cellulos... more White-rot fungi can degrade all lignocellulose components due to their potent lignin and cellulose-degrading enzymes. In this study, five white-rot fungi, Trametes versicolor, Trametes pubescens, Ganoderma adspersum, Ganoderma lipsiense, and Rigidoporus vitreus were tested for endoglucanase, laccase, urease, and glucose-6-phosphate (G6P) production when grown with malt extract and nanocellulose in the form of TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl radical) oxidized cellulose nanofibrils (CNF) and cellulose nanocrystals (CNC). Results show that temperature plays a key role in controlling the growth of all five fungi when cultured with malt extract alone. Endoglucanase activities were highest in cultures of G. adspersum and G. lipsiense and laccase activities were highest in cultures of T. versicolor and R. vitreus. Urease activities were highest in cultures of G. adspersum, G. lipsiense, and R. vitreus. Glucose-6-phosphate levels also indicate that cells were actively metabolizing glucose present in the cultures. These results show that TEMPO-oxidized CNF and CNC do not inhibit the production of specific lignocellulose enzymes by these white-rot fungi. The apparent lack of enzymatic inhibition makes TEMPO-oxidized CNF and CNC excellent candidates for future biotechnological applications in combination with the white-rot fungi studied here.
World Journal of Microbiology and Biotechnology volume, 2020
Melanins are natural biopolymers that are known to contribute to different biological processes a... more Melanins are natural biopolymers that are known to contribute to different biological processes and to protect organisms from adverse environmental conditions. During the past decade, melanins have attracted increasing attention for their use in organic semiconductors and bioelectronics, drug delivery, photoprotection and environmental bioremediation. Although considerable advances in these fields have been achieved, real-world applications of melanins are still scarce, probably due to the limited and expensive source of natural melanin. Nevertheless, recent biotechnological advances have allowed for relatively large-scale production of microbial melanins, which could replace current commercial melanin. In this review, we first describe different melanin sources and highlight the advantages and disadvantages of each production method. Our focus is on the microbial synthesis of melanins, including the methodology and mechanism of melanin formation. Applications of microbial melanins are also discussed, and an outlook on how to push the field forward is discussed.
Ammonia-oxidizing archaea (AOA) are widespread in nature and are involved in nitrification, an es... more Ammonia-oxidizing archaea (AOA) are widespread in nature and are involved in nitrification, an essential process in the global nitrogen cycle. The enzymes for ammonia oxidation and electron transport rely heavily on copper (Cu), which can be limited in nature. In this study the model soil archaeon Nitrososphaera viennensis was investigated via transcriptomic analysis to gain insight regarding possible Cu uptake mechanisms and compensation strategies when Cu becomes limiting. Upon Cu limitation, N. viennensis exhibited impaired nitrite production and thus growth, which was paralleled by downregulation of ammonia oxidation, electron transport, carbon fixation, nucleotide, and lipid biosynthesis pathway genes. Under Cu-limitation, 1547 out of 3180 detected genes were differentially expressed, with 784 genes upregulated and 763 downregulated. The most highly upregulated genes encoded proteins with a possible role in Cu binding and uptake, such as the Cu chelator and transporter CopC/D, disulfide bond oxidoreductase D (dsbD), and multicopper oxidases. While this response differs from the marine strain Nitrosopumilus maritimus, conserved sequence motifs in some of the Cu-responsive genes suggest conserved transcriptional regulation in terrestrial AOA. This study provides possible gene regulation and energy conservation mechanisms linked to Cu bioavailability and presents the first model for Cu uptake by a soil AOA.
Ammonia oxidizing archaea (AOA) inhabiting soils have a central role in the global nitrogen cycle... more Ammonia oxidizing archaea (AOA) inhabiting soils have a central role in the global nitrogen cycle. Copper (Cu) is central to many enzymes in AOA including ammonia monooxygenase (AMO), the enzyme involved in the first step of ammonia oxidation. This study explored the physiological response of the AOA soil isolate, Nitrososphaera viennensis (EN76T) to Cu-limiting conditions in order to approach its limiting threshold under laboratory conditions. The chelator TETA (1,4,8,11-tetraazacyclotetradecane N, N',N'',N'''-tetraacetic acid hydrochloride hydrate) with selective affinity for Cu2+ was used to lower bioavailable Cu2+ in culture experiments as predicted by thermodynamic speciation calculations. Results show that N. viennensis is Cu-limited at concentrations ≤10^-15 mol/L free Cu2+ compared to standard conditions (10^-12 mol/L). This Cu2+ limiting threshold is similar to pure cultures of denitrifying bacteria and other AOA and AOB inhabiting soils, freshwaters and sewage (<10^-16 mol/L), and lower than pure cultures of the marine AOA Nitrosopumilus maritimus (<10^-12.7 mol/L), which also possesses a high amount of Cu-dependent enzymes.
International Journal of Systematic and Evolutionary Microbiology , 2019
Two mesophilic, neutrophilic and aerobic marine ammonia-oxidizing archaea, designated strains NF5... more Two mesophilic, neutrophilic and aerobic marine ammonia-oxidizing archaea, designated strains NF5T and D3CT, were isolated from coastal surface water of the Northern Adriatic Sea. Cells were straight small rods 0.20–0.25 µm wide and 0.49–2.00 µm long. Strain NF5T possessed archaella as cell appendages. Glycerol dibiphytanyl glycerol tetraethers with zero to four cyclopentane moieties (GDGT-0 to GDGT-4) and crenarchaeol were the major core lipids. Menaquinone MK6 : 0 was the major respiratory quinone. Both isolates gained energy by oxidizing ammonia (NH3) to nitrite (NO2 -) and used bicarbonate as a carbon source. Strain D3CT was able use urea as a source of ammonia for energy production and growth. Addition of hydrogen peroxide (H2O2) scavengers (catalase or α-keto acids) was required to sustain growth. Optimal growth occurred between 30 and 32 °C, pH 7.1 and 7.3 and between 34 and 37‰ salinity. The cellular metal abundance ranking of both strains was Fe>Zn>Cu>Mn>Co. The genomes of strains NF5T and D3CT have a DNA G+C content of 33.4 and 33.8 mol%, respectively. Phylogenetic analyses of 16S rRNA gene sequences revealed that both strains are affiliated with the class Nitrososphaeria , sharing ~85 % 16S rRNA gene sequence identity with Nitrososphaera viennensis EN76T. The two isolates are separated by phenotypic and genotypic characteristics and are assigned to distinct species within the genus Nitrosopumilus gen. nov. according to average nucleotide identity thresholds of their closed genomes. Isolates NF5T (=JCM 32270T =NCIMB 15114T) and D3CT (=JCM 32271T =DSM 106147T =NCIMB 15115T) are type strains of the species Nitrosopumilus adriaticus sp. nov. and Nitrosopumilus piranensis sp. nov., respectively.
One sentence summary: This study gives insight into the bacterial community composition related t... more One sentence summary: This study gives insight into the bacterial community composition related to iron-cycling in suboxic marine sediments based on 16S rRNA gene pyrosequencing. ABSTRACT To gain insight into the bacterial communities involved in iron-(Fe) cycling under marine conditions, we analysed sediments with Fe-contents (0.5–1.5 wt %) from the suboxic zone at a marine site in the Skagerrak (SK) and a brackish site in the Bothnian Bay (BB) using 16S rRNA gene pyrosequencing. Several bacterial families, including Desulfobulbaceae, Desulfuromonadaceae and Pelobacteraceae and genera, including Desulfobacter and Geobacter, known to reduce Fe were detected and showed highest abundance near the Fe(III)/Fe(II) redox boundary. Additional genera with microorganisms capable of coupling fermentation to Fe-reduction, including Clostridium and Bacillus, were observed. Also, the Fe-oxidizing families Mariprofundaceae and Gallionellaceae occurred at the SK and BB sites, respectively, supporting Fe-cycling. In contrast, the sulphate (SO 4 2−) reducing bacteria Desulfococcus and Desulfobacterium were more abundant at greater depths concurring with a decrease in Fe-reducing activity. The communities revealed by pyrosequencing, thus, match the redox stratification indicated by the geochemistry, with the known Fe-reducers coinciding with the zone of Fe-reduction. Not the intensely studied model organisms, such as Geobacter spp., but rather versatile microorganisms, including sulphate reducers and possibly unknown groups appear to be important for Fe-reduction in these marine suboxic sediments.
The fate and transport of arsenic is regulated, in part, by its strong affinity for iron (hydr)ox... more The fate and transport of arsenic is regulated, in part, by its strong affinity for iron (hydr)oxides. A transition from aerobic to anaerobic conditions resulting in concomitant reduction of both As(V) and iron (hydr)oxides can thus have a pronounced influence on As partitioning. However, it is presently unclear whether As desorption under anaerobic conditions results predominantly from a transformation from As(V) to As(III) or from mineralogical changes as a consequence of iron and manganese reduction. Here, we examine desorption of both As(III) and As(V) from ferrihydrite-, goethite-, and hematite-coated sand under hydrodynamic conditions. Furthermore, to resolve the relative role of Fe(III) and/or As(V) reduction in regulating dissolved As concentrations, we also examined As desorption from ferrihydrite-and goethite-coated sands presorbed with As(V) using wild type or mutants of Shewanella sp. ANA-3, capable of Fe(III)-and/or As(V)-reduction. We reveal substantial differences in As(III) and As(V) desorption from ferrihydrite, goethite, and hematite. Despite being adsorbed to a greater extent than As(V), As(III) is desorbed more rapidly and extensively from all oxides, suggesting weaker binding of As(III) than As(V). When As(V) and Fe(III) reduction are decoupled, As(V) reduction appears to be the dominant process controlling As release. Our results also suggest the importance of appreciating physical properties of specific Fe (hydr)oxides when predicting the potential for As desorption.
Extracellular respiration of solid-phase electron acceptors in some microorganisms requires a com... more Extracellular respiration of solid-phase electron acceptors in some microorganisms requires a complex chain of multiheme ctype cytochromes that span the inner and outer membranes. In Shewanella species, MtrA, an35-kDa periplasmic decaheme c-type cytochrome, is an essential component for extracellular respiration of iron(III). The exact mechanism of electron transport has not yet been resolved, but the arrangement of the polypeptide chain may have a strong influence on the capability of the MtrA cytochrome to transport electrons. The iron hemes of MtrA are bound to its polypeptide chain via proximal (CXXCH) and distal histidine residues. In this study, we show the effects of mutating histidine residues of MtrA to arginine on protein expression and extracellular respiration using Shewanella sp. strain ANA-3 as a model organism. Individual mutations to six out of nine proximal histidines in CXXCH of MtrA led to decreased protein expression. However, distal histidine mutations resulted in various degrees of protein expression. In addition, the effects of histidine mutations on extracellular respiration were tested using ferrihydrite and current production in microbial fuel cells. These results show that proximal histidine mutants were unable to reduce ferrihydrite. Mutations to the distal histidine residues resulted in various degrees of ferrihydrite reduction. These findings indicate that mutations to the proximal histidine residues affect MtrA expression, leading to loss of extracellular respiration ability. In contrast, mutations to the distal histidine residues are less detrimental to protein expression, and extracellular respiration can proceed.
Arsenate respiration and Fe(III) reduction are important processes that influence the fate and tr... more Arsenate respiration and Fe(III) reduction are important processes that influence the fate and transport of arsenic in the environment. The goal of this study was to investigate the impact of arsenate on Fe(III) reduction using arsenate and Fe(III) reduction deficient mutants of Shewanella sp. strain ANA-3. Ferrihydrite reduction in the absence of arsenate was similar for an arsenate reduction mutant (arrA and arsC deletion strain of ANA-3) compared with wild-type ANA-3. However, the presence of arsenate adsorbed onto fer-rihydrite impeded Fe(III) reduction for the arsenate reduction mutant but not in the wild-type. In an Fe(III) reduction mutant (mtrDEF, omcA, mtrCAB null mutant of ANA-3), arsenate was reduced similarly to wild-type ANA-3 indicating the Fe(III) reduction pathway is not required for ferrihydrite-associated arsenate reduction. Expression analysis of the mtr/omc gene cluster of ANA-3 showed that omcA and mtrCAB were expressed under soluble Fe(III), ferrihydrite and arsenate growth conditions and not in aerobically grown cells. Expression of arrA was greater with ferrihydrite pre-adsorbed with arsenate relative to ferrihydrite only. Lastly, arrA and mtrA were simultaneously induced in cells shifted to anaerobic conditions and exposed to soluble Fe(III) and arsenate. These observations suggest that, unlike Fe(III), arsen-ate can co-induce operons (arr and mtr) implicated in arsenic mobilization.
In many marine surface sediments, the reduction of manganese (Mn) and iron (Fe) oxides is obscure... more In many marine surface sediments, the reduction of manganese (Mn) and iron (Fe) oxides is obscured by sulfate reduction, which is regarded as the predominant anaerobic microbial respiration process. However, many dissimilatory sulfate and sulfur reducing microorganisms are known to utilize alternative electron acceptors such as metal oxides. In this study, we tested whether sulfate and sulfur reducing bacteria are linked to metal oxide reduction based on biogeochemical modeling of porewater concentration profiles of Mn2+ and Fe2+ in Bothnian Bay (BB) and Skagerrak (SK) sediments. Steady-state modeling of Fe2+ and Mn2+ porewater profiles revealed zones of net Fe (0–9 cm BB; ∼10 and 20 cm SK) and Mn (0–5 cm BB; 2–8 cm SK) species transformations. 16S rRNA pyrosequencing analysis of the in-situ community showed that Desulfobacteraceae, Desulfuromonadaceae and Desulfobulbaceae were present in the zone of Fe-reduction of both sediments. Rhodobacteraceae were also detected at high relative abundance in both sediments. BB sediments appeared to harbor a greater diversity of potential Fe-reducers compared to SK. Additionally, when the upper 10 cm of sediment from the SK was incubated with lepidocrocite and acetate, Desulfuromonas was the dominant bacteria. Real-time quantitative polymerase chain reaction (qPCR) results showed decreasing dsrA gene copy numbers with depth coincided with decreased Fe-reduction activity. Our results support the idea that sulfur and sulfate reducing bacteria contribute to Fe-reduction in the upper centimeters of both sediments.
In this study, we analysed metagenomes along with biogeochemical profiles from Skagerrak (SK) and... more In this study, we analysed metagenomes along with biogeochemical profiles from Skagerrak (SK) and Bothnian Bay (BB) sediments, to trace the prevailing nitrogen pathways. NO 3 − was present in the top 5 cm below the sediment-water interface at both sites. NH 4 + increased with depth below 5 cm where it overlapped with the NO 3 − zone. Steady-state modelling of NO 3 − and NH 4 + porewater profiles indicates zones of net nitrogen species transformations. Bacterial protease and hydratase genes appeared to make up the bulk of total ammonification genes. Genes involved in ammonia oxidation (amo, hao), denitrification (nir, nor), dis-similatory NO 3 − reduction to NH 4 + (nfr and otr) and in both of the latter two pathways (nar, nap) were also present. Results show ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) are similarly abundant in both sediments. Also, denitrification genes appeared more abundant than DNRA genes. 16S rRNA gene analysis showed that the relative abundance of the nitrifying group Nitrosopumilales and other groups involved in nitrification and denitrification (Nitrobacter, Nitrosomonas, Nitrospira, Nitrosococcus and Nitrosomonas) appeared less abundant in SK sediments compared to BB sediments. Beggiatoa and Thiothrix 16S rRNA genes were also present, suggesting chemolithoautotrophic NO 3 − reduction to NO 2 − or NH 4 + as a possible pathway. Our results show the metabolic potential for ammonification, nitrification, DNRA and denitrification activities in North Sea and Baltic Sea sediments.
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Papers by Carolina Reyes
cytochromes that span the inner and outer membranes. In Shewanella species, MtrA, an35-kDa periplasmic decaheme
c-type cytochrome, is an essential component for extracellular respiration of iron(III). The exact mechanism of electron transport
has not yet been resolved, but the arrangement of the polypeptide chain may have a strong influence on the capability of the
MtrA cytochrome to transport electrons. The iron hemes of MtrA are bound to its polypeptide chain via proximal (CXXCH) and
distal histidine residues. In this study, we show the effects of mutating histidine residues of MtrA to arginine on protein expression
and extracellular respiration using Shewanella sp. strain ANA-3 as a model organism. Individual mutations to six out of
nine proximal histidines in CXXCH of MtrA led to decreased protein expression. However, distal histidine mutations resulted in
various degrees of protein expression. In addition, the effects of histidine mutations on extracellular respiration were tested using
ferrihydrite and current production in microbial fuel cells. These results show that proximal histidine mutants were unable
to reduce ferrihydrite. Mutations to the distal histidine residues resulted in various degrees of ferrihydrite reduction. These findings
indicate that mutations to the proximal histidine residues affect MtrA expression, leading to loss of extracellular respiration
ability. In contrast, mutations to the distal histidine residues are less detrimental to protein expression, and extracellular respiration
can proceed.
cytochromes that span the inner and outer membranes. In Shewanella species, MtrA, an35-kDa periplasmic decaheme
c-type cytochrome, is an essential component for extracellular respiration of iron(III). The exact mechanism of electron transport
has not yet been resolved, but the arrangement of the polypeptide chain may have a strong influence on the capability of the
MtrA cytochrome to transport electrons. The iron hemes of MtrA are bound to its polypeptide chain via proximal (CXXCH) and
distal histidine residues. In this study, we show the effects of mutating histidine residues of MtrA to arginine on protein expression
and extracellular respiration using Shewanella sp. strain ANA-3 as a model organism. Individual mutations to six out of
nine proximal histidines in CXXCH of MtrA led to decreased protein expression. However, distal histidine mutations resulted in
various degrees of protein expression. In addition, the effects of histidine mutations on extracellular respiration were tested using
ferrihydrite and current production in microbial fuel cells. These results show that proximal histidine mutants were unable
to reduce ferrihydrite. Mutations to the distal histidine residues resulted in various degrees of ferrihydrite reduction. These findings
indicate that mutations to the proximal histidine residues affect MtrA expression, leading to loss of extracellular respiration
ability. In contrast, mutations to the distal histidine residues are less detrimental to protein expression, and extracellular respiration
can proceed.