Sellstedt, Anita - Energy Production by Microorganisms

@article{bizjak_presence_2023,
	title = {Presence and activity of nitrogen-fixing bacteria in {Scots} pine needles in a boreal forest: a nitrogen-addition experiment},
	volume = {43},
	issn = {1758-4469},
	shorttitle = {Presence and activity of nitrogen-fixing bacteria in {Scots} pine needles in a boreal forest},
	url = {https://doi.org/10.1093/treephys/tpad048},
	doi = {10.1093/treephys/tpad048},
	abstract = {Endophytic nitrogen-fixing bacteria have been detected and isolated from the needles of conifer trees growing in North American boreal forests. Because boreal forests are nutrient-limited, these bacteria could provide an important source of nitrogen for tree species. This study aimed to determine their presence and activity in a Scandinavian boreal forest, using immunodetection of nitrogenase enzyme subunits and acetylene-reduction assays of native Scots pine (Pinus sylvestris L.) needles. The presence and rate of nitrogen fixation by endophytic bacteria were compared between control plots and fertilized plots in a nitrogen-addition experiment. In contrast to the expectation that nitrogen-fixation rates would decline in fertilized plots, as seen, for instance, with nitrogen-fixing bacteria associated with bryophytes, there was no difference in the presence or activity of nitrogen-fixing bacteria between the two treatments. The extrapolated calculated rate of nitrogen fixation relevant for the forest stand was 20 g N ha−1 year−1, which is rather low compared with Scots pine annual nitrogen use but could be important for the nitrogen-poor forest in the long term. In addition, of 13 colonies of potential nitrogen-fixing bacteria isolated from the needles on nitrogen-free media, 10 showed in vitro nitrogen fixation. In summary, 16S rRNA sequencing identified the species as belonging to the genera Bacillus, Variovorax, Novosphingobium, Sphingomonas, Microbacterium and Priestia, which was confirmed by Illumina whole-genome sequencing. Our results confirm the presence of endophytic nitrogen-fixing bacteria in Scots pine needles and suggest that they could be important for the long-term nitrogen budget of the Scandinavian boreal forest.},
	number = {8},
	urldate = {2023-08-21},
	journal = {Tree Physiology},
	author = {Bizjak, Tinkara and Sellstedt, Anita and Gratz, Regina and Nordin, Annika},
	month = aug,
	year = {2023},
	pages = {1354--1364},
}

Endophytic nitrogen-fixing bacteria have been detected and isolated from the needles of conifer trees growing in North American boreal forests. Because boreal forests are nutrient-limited, these bacteria could provide an important source of nitrogen for tree species. This study aimed to determine their presence and activity in a Scandinavian boreal forest, using immunodetection of nitrogenase enzyme subunits and acetylene-reduction assays of native Scots pine (Pinus sylvestris L.) needles. The presence and rate of nitrogen fixation by endophytic bacteria were compared between control plots and fertilized plots in a nitrogen-addition experiment. In contrast to the expectation that nitrogen-fixation rates would decline in fertilized plots, as seen, for instance, with nitrogen-fixing bacteria associated with bryophytes, there was no difference in the presence or activity of nitrogen-fixing bacteria between the two treatments. The extrapolated calculated rate of nitrogen fixation relevant for the forest stand was 20 g N ha−1 year−1, which is rather low compared with Scots pine annual nitrogen use but could be important for the nitrogen-poor forest in the long term. In addition, of 13 colonies of potential nitrogen-fixing bacteria isolated from the needles on nitrogen-free media, 10 showed in vitro nitrogen fixation. In summary, 16S rRNA sequencing identified the species as belonging to the genera Bacillus, Variovorax, Novosphingobium, Sphingomonas, Microbacterium and Priestia, which was confirmed by Illumina whole-genome sequencing. Our results confirm the presence of endophytic nitrogen-fixing bacteria in Scots pine needles and suggest that they could be important for the long-term nitrogen budget of the Scandinavian boreal forest.

@article{nzayisenga_metabolomic_2021,
	title = {Metabolomic {Study} of {Heterotrophically} {Grown} {Chlorella} sp. {Isolated} from {Wastewater} in {Northern} {Sweden}},
	volume = {26},
	issn = {1420-3049},
	url = {https://www.mdpi.com/1420-3049/26/9/2410},
	doi = {10/gj6zk8},
	abstract = {There are numerous strains of Chlorella with a corresponding variety of metabolic pathways. A strain we previously isolated from wastewater in northern Sweden can grow heterotrophically as well as autotrophically in light and has higher lipid contents under heterotrophic growth conditions. The aims of the present study were to characterize metabolic changes associated with the higher lipid contents in order to enhance our understanding of lipid production in microalgae and potentially identify new compounds with utility in sustainable development. Inter alia, the amino acids glutamine and lysine were 7-fold more abundant under heterotrophic conditions, the key metabolic intermediate alpha-ketoglutarate was more abundant under heterotrophic conditions with glucose, and maltose was more abundant under heterotrophic conditions with glycerol than under autotrophic conditions. The metabolite 3-hydroxy-butyric acid, the direct precursor of the biodegradable plastic PHB (poly-3-hydroxy-butyric acid), was also more abundant under heterotrophic conditions. Our metabolomic analysis has provided new insights into the alga’s lipid production pathways and identified metabolites with potential use in sustainable development, such as the production of renewable, biodegradable plastics, cosmetics, and nutraceuticals, with reduced pollution and improvements in both ecological and human health.},
	language = {en},
	number = {9},
	urldate = {2021-06-03},
	journal = {Molecules},
	author = {Nzayisenga, Jean Claude and Sellstedt, Anita},
	month = apr,
	year = {2021},
	pages = {2410},
}

There are numerous strains of Chlorella with a corresponding variety of metabolic pathways. A strain we previously isolated from wastewater in northern Sweden can grow heterotrophically as well as autotrophically in light and has higher lipid contents under heterotrophic growth conditions. The aims of the present study were to characterize metabolic changes associated with the higher lipid contents in order to enhance our understanding of lipid production in microalgae and potentially identify new compounds with utility in sustainable development. Inter alia, the amino acids glutamine and lysine were 7-fold more abundant under heterotrophic conditions, the key metabolic intermediate alpha-ketoglutarate was more abundant under heterotrophic conditions with glucose, and maltose was more abundant under heterotrophic conditions with glycerol than under autotrophic conditions. The metabolite 3-hydroxy-butyric acid, the direct precursor of the biodegradable plastic PHB (poly-3-hydroxy-butyric acid), was also more abundant under heterotrophic conditions. Our metabolomic analysis has provided new insights into the alga’s lipid production pathways and identified metabolites with potential use in sustainable development, such as the production of renewable, biodegradable plastics, cosmetics, and nutraceuticals, with reduced pollution and improvements in both ecological and human health.

@article{harinikumar_bioethanol_2020,
	title = {Bioethanol production from four abundant {Indian} agricultural wastes},
	volume = {11},
	issn = {1759-7269, 1759-7277},
	url = {https://www.tandfonline.com/doi/full/10.1080/17597269.2017.1387744},
	doi = {10.1080/17597269.2017.1387744},
	language = {en},
	number = {5},
	urldate = {2021-06-07},
	journal = {Biofuels},
	author = {Harinikumar, K. M. and Kudahettige-Nilsson, R. L. and Devadas, A. and Holmgren, M. and Sellstedt, A.},
	month = jul,
	year = {2020},
	pages = {607--613},
}

@article{herrera-belaroussi_candidatus_2020,
	title = {Candidatus {Frankia} nodulisporulans sp. nov., an {Alnus} glutinosa-infective {Frankia} species unable to grow in pure culture and able to sporulate in-planta},
	volume = {43},
	issn = {07232020},
	url = {https://linkinghub.elsevier.com/retrieve/pii/S0723202020300898},
	doi = {10/gjdt2w},
	language = {en},
	number = {6},
	urldate = {2021-06-07},
	journal = {Systematic and Applied Microbiology},
	author = {Herrera-Belaroussi, Aude and Normand, Philippe and Pawlowski, Katharina and Fernandez, Maria P. and Wibberg, Daniel and Kalinowski, Jörn and Brachmann, Andreas and Berckx, Fede and Lee, Natuschka and Blom, Jochen and Pozzi, Adrien C. and Fournier, Pascale and Bethencourt, Lorine and Dubost, Audrey and Abrouk, Danis and Sellstedt, Anita},
	month = nov,
	year = {2020},
	pages = {126134},
}

@article{nzayisenga_effects_2020,
	title = {Effects of light intensity on growth and lipid production in microalgae grown in wastewater},
	volume = {13},
	issn = {1754-6834},
	url = {https://biotechnologyforbiofuels.biomedcentral.com/articles/10.1186/s13068-019-1646-x},
	doi = {10.1186/s13068-019-1646-x},
	abstract = {Abstract
            
              Background
              
                Cultivation of microalgae in wastewater could significantly contribute to wastewater treatment, biodiesel production, and thus the transition to renewable energy. However, more information on effects of environmental factors, including light intensity, on their growth and composition (particularly fatty acid contents) is required. Therefore, we investigated the biomass and fatty acid production of four microalgal species, isolated in the Northern hemisphere and grown at three light intensities (50, 150 and 300 μE m
                −2
                s
                −1
                ).
              
            
            
              Results
              
                Increases in light intensities resulted in higher biomass of all four species and, importantly, raised fatty acid contents of both
                Desmodesmus
                sp. and
                Scenedesmus obliquus
                . Fourier-transform IR spectrometry analysis showed that the increases in fatty acid content were associated with reductions in protein, but not carbohydrate, contents. Assessment of fatty acid composition revealed that increasing light intensity led to higher and lower contents of oleic (18:1) and linolenic (18:3) acids, respectively. The microalgae consumed more than 75\% of the nitrogen and phosphorus present in the wastewater used as growth medium.
              
            
            
              Conclusion
              The results show the importance of optimizing light intensities to improve fatty acid production by microalgae and their quality as sources of biodiesel. In addition, increase in fatty acid content is associated with decrease in protein content.},
	language = {en},
	number = {1},
	urldate = {2021-06-07},
	journal = {Biotechnology for Biofuels},
	author = {Nzayisenga, Jean Claude and Farge, Xavier and Groll, Sophia Leticia and Sellstedt, Anita},
	month = dec,
	year = {2020},
	pages = {4},
}

Abstract Background Cultivation of microalgae in wastewater could significantly contribute to wastewater treatment, biodiesel production, and thus the transition to renewable energy. However, more information on effects of environmental factors, including light intensity, on their growth and composition (particularly fatty acid contents) is required. Therefore, we investigated the biomass and fatty acid production of four microalgal species, isolated in the Northern hemisphere and grown at three light intensities (50, 150 and 300 μE m −2 s −1 ). Results Increases in light intensities resulted in higher biomass of all four species and, importantly, raised fatty acid contents of both Desmodesmus sp. and Scenedesmus obliquus . Fourier-transform IR spectrometry analysis showed that the increases in fatty acid content were associated with reductions in protein, but not carbohydrate, contents. Assessment of fatty acid composition revealed that increasing light intensity led to higher and lower contents of oleic (18:1) and linolenic (18:3) acids, respectively. The microalgae consumed more than 75% of the nitrogen and phosphorus present in the wastewater used as growth medium. Conclusion The results show the importance of optimizing light intensities to improve fatty acid production by microalgae and their quality as sources of biodiesel. In addition, increase in fatty acid content is associated with decrease in protein content.

@article{kawde_more_2020,
	title = {More than protection: the function of {TiO} $_{\textrm{2}}$ interlayers in hematite functionalized {Si} photoanodes},
	volume = {22},
	issn = {1463-9076, 1463-9084},
	shorttitle = {More than protection},
	url = {http://xlink.rsc.org/?DOI=D0CP04280C},
	doi = {10/gjdpf7},
	abstract = {Signature of performance-enhancing oxygen vacancies in the mesoporous TiO
              2
               interlayer of a hematite functionalized Si microwire photoanode revealed by hard energy X-ray spectroscopy.
            
          , 
            
              Worldwide significant efforts are ongoing to develop devices that store solar energy as fuels. In one such approach, solar energy is absorbed by semiconductors and utilized directly by catalysts at their surfaces to split water into H
              2
              and O
              2
              . To protect the semiconductors in these photo-electrochemical cells (PEC) from corrosion, frequently thin TiO
              2
              interlayers are applied. Employing a well-performing photoanode comprised of 1-D n-Si microwires (MWs) covered with a mesoporous (mp) TiO
              2
              interlayer fabricated by solution processing and functionalized with α-Fe
              2
              O
              3
              nanorods, we studied here the function of this TiO
              2
              interlayer by high-energy resolution fluorescence detected X-ray absorption near edge structure (HERFD-XANES) spectroscopy, along with X-ray emission spectroscopy (XES) and standard characterization techniques. Our data reveal that the TiO
              2
              interlayer not only protects the n-Si MW surface from corrosion, but that it also acts as a template for the hydrothermal growth of α-Fe
              2
              O
              3
              nanorods and improves the photocatalytic efficiency. We show that the latter effect correlates with the presence of stable oxygen vacancies at the interface between mp-TiO
              2
              and α-Fe
              2
              O
              3
              , which act as electron traps and thereby substantially reduce the charge recombination rate at the hematite surface.},
	language = {en},
	number = {48},
	urldate = {2021-06-07},
	journal = {Physical Chemistry Chemical Physics},
	author = {Kawde, Anurag and Annamalai, Alagappan and Sellstedt, Anita and Uhlig, Jens and Wågberg, Thomas and Glatzel, Pieter and Messinger, Johannes},
	year = {2020},
	pages = {28459--28467},
}

Signature of performance-enhancing oxygen vacancies in the mesoporous TiO 2  interlayer of a hematite functionalized Si microwire photoanode revealed by hard energy X-ray spectroscopy. , Worldwide significant efforts are ongoing to develop devices that store solar energy as fuels. In one such approach, solar energy is absorbed by semiconductors and utilized directly by catalysts at their surfaces to split water into H 2 and O 2 . To protect the semiconductors in these photo-electrochemical cells (PEC) from corrosion, frequently thin TiO 2 interlayers are applied. Employing a well-performing photoanode comprised of 1-D n-Si microwires (MWs) covered with a mesoporous (mp) TiO 2 interlayer fabricated by solution processing and functionalized with α-Fe 2 O 3 nanorods, we studied here the function of this TiO 2 interlayer by high-energy resolution fluorescence detected X-ray absorption near edge structure (HERFD-XANES) spectroscopy, along with X-ray emission spectroscopy (XES) and standard characterization techniques. Our data reveal that the TiO 2 interlayer not only protects the n-Si MW surface from corrosion, but that it also acts as a template for the hydrothermal growth of α-Fe 2 O 3 nanorods and improves the photocatalytic efficiency. We show that the latter effect correlates with the presence of stable oxygen vacancies at the interface between mp-TiO 2 and α-Fe 2 O 3 , which act as electron traps and thereby substantially reduce the charge recombination rate at the hematite surface.

@article{nzayisenga_screening_2020,
	title = {Screening {Suitability} of {Northern} {Hemisphere} {Algal} {Strains} for {Heterotrophic} {Cultivation} and {Fatty} {Acid} {Methyl} {Ester} {Production}},
	volume = {25},
	issn = {1420-3049},
	url = {https://www.mdpi.com/1420-3049/25/9/2107},
	doi = {10.3390/molecules25092107},
	abstract = {Rapid rises in atmospheric CO2 levels derived from fossil fuel combustion are imposing urgent needs for renewable substitutes. One environmentally friendly alternative is biodiesel produced from suitable microalgal fatty acids. Algal strains normally grow photoautotrophically, but this is problematic in Northern areas because of the light limitations for much of the year. Mixotrophic and particularly heterotrophic strains could be valuable, especially if they can be cultivated in municipal wastewater with contents of nutrients such as nitrogen and phosphorous that should be reduced before release into receiving water. Thus, the aim of this study was to screen for microalgal strains suitable for heterotrophic cultivation with a cheap carbon source (glycerol) for biodiesel production in Nordic, and other high-latitude, countries. One of the examined strains, a Desmodesmus sp. strain designated 2-6, accumulated biomass at similar rates in heterotrophic conditions with 40 mM glycerol as in autotrophic conditions. Furthermore, in heterotrophic conditions it produced more fatty acids, and ca. 50\% more C18:1 fatty acids, as well as showing a significant decrease in C18:3 fatty acids, all of which are highly desirable features for biodiesel production.},
	language = {en},
	number = {9},
	urldate = {2021-06-07},
	journal = {Molecules},
	author = {Nzayisenga, Jean Claude and Niemi, Calle and Ferro, Lorenza and Gorzsas, Andras and Gentili, Francesco G. and Funk, Christiane and Sellstedt, Anita},
	month = apr,
	year = {2020},
	pages = {2107},
}

Rapid rises in atmospheric CO2 levels derived from fossil fuel combustion are imposing urgent needs for renewable substitutes. One environmentally friendly alternative is biodiesel produced from suitable microalgal fatty acids. Algal strains normally grow photoautotrophically, but this is problematic in Northern areas because of the light limitations for much of the year. Mixotrophic and particularly heterotrophic strains could be valuable, especially if they can be cultivated in municipal wastewater with contents of nutrients such as nitrogen and phosphorous that should be reduced before release into receiving water. Thus, the aim of this study was to screen for microalgal strains suitable for heterotrophic cultivation with a cheap carbon source (glycerol) for biodiesel production in Nordic, and other high-latitude, countries. One of the examined strains, a Desmodesmus sp. strain designated 2-6, accumulated biomass at similar rates in heterotrophic conditions with 40 mM glycerol as in autotrophic conditions. Furthermore, in heterotrophic conditions it produced more fatty acids, and ca. 50% more C18:1 fatty acids, as well as showing a significant decrease in C18:3 fatty acids, all of which are highly desirable features for biodiesel production.

@article{kawde_microstructured_2019,
	title = {A microstructured p-{Si} photocathode outcompetes {Pt} as a counter electrode to hematite in photoelectrochemical water splitting},
	volume = {48},
	issn = {1477-9226, 1477-9234},
	url = {http://xlink.rsc.org/?DOI=C8DT03653E},
	doi = {10.1039/C8DT03653E},
	abstract = {Herein we demonstrate that an earth-abundant semiconductor photocathode (p-Si/TiO
              2
              /NiO
              x
              ) out-competes rare and expensive Pt as counter electrode to Fe-oxide for overall photoelectrochemical water splitting.
            
          , 
            
              Herein, we communicate about an Earth-abundant semiconductor photocathode (p-Si/TiO
              2
              /NiO
              x
              ) as an alternative for the rare and expensive Pt as a counter electrode for overall photoelectrochemical water splitting. The proposed photoelectrochemical (PEC) water-splitting device mimics the “Z”-scheme observed in natural photosynthesis by combining two photoelectrodes in a parallel-illumination mode. A nearly 60\% increase in the photocurrent density (
              J
              ph
              ) for pristine α-Fe
              2
              O
              3
              and a 77\% increase in the applied bias photocurrent efficiency (ABPE) were achieved by replacing the conventionally used Pt cathode with an efficient, cost effective p-Si/TiO
              2
              /NiO
              x
              photocathode under parallel illumination. The resulting photocurrent density of 1.26 mA cm
              −2
              at 1.23
              V
              RHE
              represents a new record performance for hydrothermally grown pristine α-Fe
              2
              O
              3
              nanorod photoanodes in combination with a photocathode, which opens the prospect for further improvement by doping α-Fe
              2
              O
              3
              or by its decoration with co-catalysts. Electrochemical impedance spectroscopy measurements suggest that this significant performance increase is due to the enhancement of the space-charge field in α-Fe
              2
              O
              3
              .},
	language = {en},
	number = {4},
	urldate = {2021-06-07},
	journal = {Dalton Transactions},
	author = {Kawde, Anurag and Annamalai, Alagappan and Sellstedt, Anita and Glatzel, Pieter and Wågberg, Thomas and Messinger, Johannes},
	year = {2019},
	pages = {1166--1170},
}

Herein we demonstrate that an earth-abundant semiconductor photocathode (p-Si/TiO 2 /NiO x ) out-competes rare and expensive Pt as counter electrode to Fe-oxide for overall photoelectrochemical water splitting. , Herein, we communicate about an Earth-abundant semiconductor photocathode (p-Si/TiO 2 /NiO x ) as an alternative for the rare and expensive Pt as a counter electrode for overall photoelectrochemical water splitting. The proposed photoelectrochemical (PEC) water-splitting device mimics the “Z”-scheme observed in natural photosynthesis by combining two photoelectrodes in a parallel-illumination mode. A nearly 60% increase in the photocurrent density ( J ph ) for pristine α-Fe 2 O 3 and a 77% increase in the applied bias photocurrent efficiency (ABPE) were achieved by replacing the conventionally used Pt cathode with an efficient, cost effective p-Si/TiO 2 /NiO x photocathode under parallel illumination. The resulting photocurrent density of 1.26 mA cm −2 at 1.23 V RHE represents a new record performance for hydrothermally grown pristine α-Fe 2 O 3 nanorod photoanodes in combination with a photocathode, which opens the prospect for further improvement by doping α-Fe 2 O 3 or by its decoration with co-catalysts. Electrochemical impedance spectroscopy measurements suggest that this significant performance increase is due to the enhancement of the space-charge field in α-Fe 2 O 3 .

@article{nzayisenga_mixotrophic_2018,
	title = {Mixotrophic and heterotrophic production of lipids and carbohydrates by a locally isolated microalga using wastewater as a growth medium},
	volume = {257},
	issn = {09608524},
	url = {https://linkinghub.elsevier.com/retrieve/pii/S0960852418302827},
	doi = {10.1016/j.biortech.2018.02.085},
	language = {en},
	urldate = {2021-06-07},
	journal = {Bioresource Technology},
	author = {Nzayisenga, Jean Claude and Eriksson, Karolina and Sellstedt, Anita},
	month = jun,
	year = {2018},
	pages = {260--265},
}

@article{kawde_photo-electrochemical_2018,
	title = {Photo-electrochemical hydrogen production from neutral phosphate buffer and seawater using micro-structured p-{Si} photo-electrodes functionalized by solution-based methods},
	volume = {2},
	issn = {2398-4902},
	url = {http://xlink.rsc.org/?DOI=C8SE00291F},
	doi = {10.1039/C8SE00291F},
	abstract = {Micro-structured p-Si/TiO
              2
              /NiO
              x
              allows for efficient photoelectrochemical H
              2
              production from seawater.
            
          , 
            
              Solar fuels such as H
              2
              generated from sunlight and seawater using earth-abundant materials are expected to be a crucial component of a next generation renewable energy mix. We herein report a systematic analysis of the photo-electrochemical performance of TiO
              2
              coated, microstructured p-Si photo-electrodes (p-Si/TiO
              2
              ) that were functionalized with CoO
              x
              and NiO
              x
              for H
              2
              generation. These photocathodes were synthesized from commercial p-Si wafers employing wet chemical methods. In neutral phosphate buffer and standard 1 sun illumination, the p-Si/TiO
              2
              /NiO
              x
              photoelectrode showed a photocurrent density of −1.48 mA cm
              −2
              at zero bias (0 V
              RHE
              ), which was three times and 15 times better than the photocurrent densities of p-Si/TiO
              2
              /CoO
              x
              and p-Si/TiO
              2
              , respectively. No decline in activity was observed over a five hour test period, yielding a Faradaic efficiency of 96\% for H
              2
              production. Based on the electrochemical characterizations and the high energy resolution fluorescence detected X-ray absorption near edge structure (HERFD-XANES) and emission spectroscopy measurements performed at the Ti Kα
              1
              fluorescence line, the superior performance of the p-Si/TiO
              2
              /NiO
              x
              photoelectrode was attributed to improved charge transfer properties induced by the NiO
              x
              coating on the protective TiO
              2
              layer, in combination with a higher catalytic activity of NiO
              x
              for H
              2
              -evolution. Moreover, we report here an excellent photo-electrochemical performance of p-Si/TiO
              2
              /NiO
              x
              photoelectrode in corrosive artificial seawater (pH 8.4) with an unprecedented photocurrent density of 10 mA cm
              −2
              at an applied potential of −0.7 V
              RHE
              , and of 20 mA cm
              −2
              at −0.9 V
              RHE
              . The applied bias photon-to-current conversion efficiency (ABPE) at −0.7 V
              RHE
              and 10 mA cm
              −2
              was found to be 5.1\%.},
	language = {en},
	number = {10},
	urldate = {2021-06-07},
	journal = {Sustainable Energy \& Fuels},
	author = {Kawde, Anurag and Annamalai, Alagappan and Amidani, Lucia and Boniolo, Manuel and Kwong, Wai Ling and Sellstedt, Anita and Glatzel, Pieter and Wågberg, Thomas and Messinger, Johannes},
	year = {2018},
	pages = {2215--2223},
}

Micro-structured p-Si/TiO 2 /NiO x allows for efficient photoelectrochemical H 2 production from seawater. , Solar fuels such as H 2 generated from sunlight and seawater using earth-abundant materials are expected to be a crucial component of a next generation renewable energy mix. We herein report a systematic analysis of the photo-electrochemical performance of TiO 2 coated, microstructured p-Si photo-electrodes (p-Si/TiO 2 ) that were functionalized with CoO x and NiO x for H 2 generation. These photocathodes were synthesized from commercial p-Si wafers employing wet chemical methods. In neutral phosphate buffer and standard 1 sun illumination, the p-Si/TiO 2 /NiO x photoelectrode showed a photocurrent density of −1.48 mA cm −2 at zero bias (0 V RHE ), which was three times and 15 times better than the photocurrent densities of p-Si/TiO 2 /CoO x and p-Si/TiO 2 , respectively. No decline in activity was observed over a five hour test period, yielding a Faradaic efficiency of 96% for H 2 production. Based on the electrochemical characterizations and the high energy resolution fluorescence detected X-ray absorption near edge structure (HERFD-XANES) and emission spectroscopy measurements performed at the Ti Kα 1 fluorescence line, the superior performance of the p-Si/TiO 2 /NiO x photoelectrode was attributed to improved charge transfer properties induced by the NiO x coating on the protective TiO 2 layer, in combination with a higher catalytic activity of NiO x for H 2 -evolution. Moreover, we report here an excellent photo-electrochemical performance of p-Si/TiO 2 /NiO x photoelectrode in corrosive artificial seawater (pH 8.4) with an unprecedented photocurrent density of 10 mA cm −2 at an applied potential of −0.7 V RHE , and of 20 mA cm −2 at −0.9 V RHE . The applied bias photon-to-current conversion efficiency (ABPE) at −0.7 V RHE and 10 mA cm −2 was found to be 5.1%.

@article{kudahettige_nilsson_adaptability_2016,
	title = {Adaptability of {Trametes} versicolor to the lignocellulosic inhibitors furfural, {HMF}, phenol and levulinic acid during ethanol fermentation},
	volume = {90},
	issn = {09619534},
	url = {https://linkinghub.elsevier.com/retrieve/pii/S0961953416300927},
	doi = {10/f3pk4h},
	language = {en},
	urldate = {2021-06-07},
	journal = {Biomass and Bioenergy},
	author = {Kudahettige Nilsson, Rasika L. and Holmgren, Marie and Madavi, Batol and Nilsson, Robert T. and Sellstedt, Anita},
	month = jul,
	year = {2016},
	pages = {95--100},
}

@article{pawar_expression_2016,
	title = {Expression of fungal acetyl xylan esterase in \textit{{Arabidopsis} thaliana} improves saccharification of stem lignocellulose},
	volume = {14},
	issn = {14677644},
	url = {http://doi.wiley.com/10.1111/pbi.12393},
	doi = {10.1111/pbi.12393},
	language = {en},
	number = {1},
	urldate = {2021-06-07},
	journal = {Plant Biotechnology Journal},
	author = {Pawar, Prashant Mohan-Anupama and Derba-Maceluch, Marta and Chong, Sun-Li and Gómez, Leonardo D. and Miedes, Eva and Banasiak, Alicja and Ratke, Christine and Gaertner, Cyril and Mouille, Grégory and McQueen-Mason, Simon J. and Molina, Antonio and Sellstedt, Anita and Tenkanen, Maija and Mellerowicz, Ewa J.},
	month = jan,
	year = {2016},
	pages = {387--397},
}

@incollection{clark_biomassbased_2015,
	edition = {1},
	title = {Biomass‐{Based} {Energy} {Production}},
	isbn = {978-1-118-71448-5 978-1-118-71447-8},
	url = {https://onlinelibrary.wiley.com/doi/abs/10.1002/9781118714478.ch7},
	abstract = {Today, bioenergy is a practical and increasingly widely available option for heating that is being adopted by many industries and households that are looking to use more sustainable energy sources. Biomass-derived fuels may be solids, liquids, or gases. They can be classified based on the processes used in their production, which include physical upgrading, microbiological processes, thermochemical processes, and chemical processes. The production of solid fuels from renewable resources has become more important due to the growing global demand for energy and environmental concerns. Various microbial processes can be exploited to utilize energy that has been stored in biomass by photosynthesis. Methanol, ethanol, and butanol are liquid biofuels that can be synthesized from biomass and used in both four-stroke gasoline and diesel engines. These alcohols can be prepared from sugarcane, sugar beet, wheat, barley, corn, switch grass, agricultural residues, wood, and many other industrial wastes},
	language = {en},
	urldate = {2021-06-07},
	booktitle = {Introduction to {Chemicals} from {Biomass}},
	publisher = {Wiley},
	author = {Arshadi, Mehrdad and Sellstedt, Anita},
	editor = {Clark, James and Deswarte, Fabien},
	month = feb,
	year = {2015},
	doi = {10.1002/9781118714478.ch7},
	pages = {249--284},
}

Today, bioenergy is a practical and increasingly widely available option for heating that is being adopted by many industries and households that are looking to use more sustainable energy sources. Biomass-derived fuels may be solids, liquids, or gases. They can be classified based on the processes used in their production, which include physical upgrading, microbiological processes, thermochemical processes, and chemical processes. The production of solid fuels from renewable resources has become more important due to the growing global demand for energy and environmental concerns. Various microbial processes can be exploited to utilize energy that has been stored in biomass by photosynthesis. Methanol, ethanol, and butanol are liquid biofuels that can be synthesized from biomass and used in both four-stroke gasoline and diesel engines. These alcohols can be prepared from sugarcane, sugar beet, wheat, barley, corn, switch grass, agricultural residues, wood, and many other industrial wastes

@article{pujic_genome_2015,
	title = {Genome {Sequence} of the {Atypical} {Symbiotic} {Frankia} {R43} {Strain}, a {Nitrogen}-{Fixing} and {Hydrogen}-{Producing} {Actinobacterium}},
	volume = {3},
	issn = {2169-8287 (Print)},
	url = {https://www.ncbi.nlm.nih.gov/pubmed/26607894},
	doi = {10.1128/genomeA.01387-15},
	abstract = {Frankia strain R43 is a nitrogen-fixing and hydrogen-producing symbiotic actinobacterium that was isolated from nodules of Casuarina cunninghamiana but infects only Elaeagnaceae. This communication reports the genome of the strain R43 and provides insights into the microbe genomics and physiological potentials.},
	language = {en},
	number = {6},
	urldate = {2021-06-07},
	journal = {Genome Announc},
	author = {Pujic, P. and Bolotin, A. and Fournier, P. and Sorokin, A. and Lapidus, A. and Richau, K. H. and Briolay, J. and Mebarki, F. and Normand, P. and Sellstedt, A.},
	month = nov,
	year = {2015},
	note = {Edition: 2015/11/27},
}

Frankia strain R43 is a nitrogen-fixing and hydrogen-producing symbiotic actinobacterium that was isolated from nodules of Casuarina cunninghamiana but infects only Elaeagnaceae. This communication reports the genome of the strain R43 and provides insights into the microbe genomics and physiological potentials.

@article{sellstedt_aspects_2013,
	title = {Aspects of nitrogen-fixing \textit{{Actinobacteria}} , in particular free-living and symbiotic \textit{{Frankia}}},
	volume = {342},
	issn = {03781097},
	url = {https://academic.oup.com/femsle/article-lookup/doi/10.1111/1574-6968.12116},
	doi = {10/f23gh6},
	language = {en},
	number = {2},
	urldate = {2021-06-08},
	journal = {FEMS Microbiology Letters},
	author = {Sellstedt, Anita and Richau, Kerstin H.},
	month = may,
	year = {2013},
	pages = {179--186},
}

@article{kudahettige_characterization_2012,
	title = {Characterization of {Bioethanol} {Production} from {Hexoses} and {Xylose} by the {White} {Rot} {Fungus} {Trametes} versicolor},
	volume = {5},
	issn = {1939-1234, 1939-1242},
	url = {http://link.springer.com/10.1007/s12155-011-9119-5},
	doi = {10/dbhx8z},
	language = {en},
	number = {2},
	urldate = {2021-06-08},
	journal = {BioEnergy Research},
	author = {Kudahettige, Rasika L. and Holmgren, Marie and Imerzeel, Peter and Sellstedt, Anita},
	month = jun,
	year = {2012},
	pages = {277--285},
}

@article{zackrisson_nitrogen_2009,
	title = {Nitrogen fixation in mixed {Hylocomium} splendens moss communities},
	volume = {160},
	issn = {0029-8549, 1432-1939},
	url = {http://link.springer.com/10.1007/s00442-009-1299-8},
	doi = {10/csd825},
	language = {en},
	number = {2},
	urldate = {2021-06-08},
	journal = {Oecologia},
	author = {Zackrisson, O. and DeLuca, T. H. and Gentili, F. and Sellstedt, A. and Jäderlund, A.},
	month = may,
	year = {2009},
	pages = {309--319},
}

@article{leul_phylogeny_2009,
	title = {The phylogeny of uptake hydrogenases in {Frankia}},
	volume = {12},
	issn = {1618-1905},
	doi = {10.2436/20.1501.01.78},
	abstract = {Uptake hydrogenase is an enzyme that is beneficial for nitrogen fixation in bacteria. Recent studies have shown that Frankia sp. has two sets of uptake hydrogenase genes, organized in synton 1 and synton 2. In the present study, phylogenetic analysis of the structural subunits of hydrogenase syntons 1 and 2 showed a distinct clustering pattern between the proteins of Frankia strains that were isolated from different host plants and non-Frankia organisms. The structural subunits of hydrogenase synton 1 of Frankia sp. CpI1, Frankia alni ACN14a, and F. alni AvCI1 were grouped together while those of Frankia spp. CcI3, KB5, UGL140104, and UGL011102 formed another group. The structural subunits of hydrogenase synton 2 of F. alni ACN14a and Frankia spp. CcI3 and BCU110501 grouped together, but those of Frankia spp. KB5 and CpI1, F. alni ArI3, and F. alniAvCI1 comprised a separate group. The structural subunits of hydrogenase syntons 1 and 2 of Frankia sp. EAN1pec were more closely related to those of non-Frankia bacteria, i.e., Streptomyces avermitilis and Anaeromyxobacter sp., respectively, than to those of other Frankia strains, suggesting the occurrence of lateral gene transfer between these organisms. In addition, the accessory Hyp proteins of hydrogenase syntons 1 and 2 of F. alni ACN14a and Frankia sp. CcI3 were shown to be phylogenetically more related to each other than to those of Frankia EAN1pec.},
	language = {eng},
	number = {1},
	journal = {International Microbiology: The Official Journal of the Spanish Society for Microbiology},
	author = {Leul, Melakeselam and Normand, Philippe and Sellstedt, Anita},
	month = mar,
	year = {2009},
	pmid = {19440980},
	keywords = {Bacterial Proteins, Frankia, Genome, Bacterial, Geography, Hydroxyproline, Nitrogen Fixation, Oxidoreductases, Phylogeny},
	pages = {23--28},
}

Uptake hydrogenase is an enzyme that is beneficial for nitrogen fixation in bacteria. Recent studies have shown that Frankia sp. has two sets of uptake hydrogenase genes, organized in synton 1 and synton 2. In the present study, phylogenetic analysis of the structural subunits of hydrogenase syntons 1 and 2 showed a distinct clustering pattern between the proteins of Frankia strains that were isolated from different host plants and non-Frankia organisms. The structural subunits of hydrogenase synton 1 of Frankia sp. CpI1, Frankia alni ACN14a, and F. alni AvCI1 were grouped together while those of Frankia spp. CcI3, KB5, UGL140104, and UGL011102 formed another group. The structural subunits of hydrogenase synton 2 of F. alni ACN14a and Frankia spp. CcI3 and BCU110501 grouped together, but those of Frankia spp. KB5 and CpI1, F. alni ArI3, and F. alniAvCI1 comprised a separate group. The structural subunits of hydrogenase syntons 1 and 2 of Frankia sp. EAN1pec were more closely related to those of non-Frankia bacteria, i.e., Streptomyces avermitilis and Anaeromyxobacter sp., respectively, than to those of other Frankia strains, suggesting the occurrence of lateral gene transfer between these organisms. In addition, the accessory Hyp proteins of hydrogenase syntons 1 and 2 of F. alni ACN14a and Frankia sp. CcI3 were shown to be phylogenetically more related to each other than to those of Frankia EAN1pec.

@article{holmgren_identification_2008,
	title = {Identification of white-rot and soft-rot fungi increasing ethanol production from spent sulfite liquor in co-culture with \textit{{Saccharomyces} cerevisiae}},
	volume = {105},
	issn = {13645072, 13652672},
	url = {http://doi.wiley.com/10.1111/j.1365-2672.2008.03724.x},
	doi = {10/cb236h},
	language = {en},
	number = {1},
	urldate = {2021-06-10},
	journal = {Journal of Applied Microbiology},
	author = {Holmgren, M. and Sellstedt, A.},
	month = jul,
	year = {2008},
	pages = {134--140},
}

@article{deluca_ecosystem_2007,
	title = {Ecosystem controls on nitrogen fixation in boreal feather moss communities},
	volume = {152},
	issn = {0029-8549, 1432-1939},
	url = {http://link.springer.com/10.1007/s00442-006-0626-6},
	doi = {10/c2tmkt},
	language = {en},
	number = {1},
	urldate = {2021-06-10},
	journal = {Oecologia},
	author = {DeLuca, Thomas H. and Zackrisson, Olle and Gentili, Francesco and Sellstedt, Anita and Nilsson, Marie-Charlotte},
	month = apr,
	year = {2007},
	pages = {121--130},
}

@article{sellstedt_frankia_2007,
	title = {Frankia ? the friendly bacteria ? infecting actinorhizal plants},
	volume = {130},
	issn = {0031-9317, 1399-3054},
	shorttitle = {Frankia ?},
	url = {http://doi.wiley.com/10.1111/j.1399-3054.2007.00932.x},
	doi = {10/cv28q8},
	language = {en},
	number = {3},
	urldate = {2021-06-10},
	journal = {Physiologia Plantarum},
	author = {Sellstedt, Anita and Normand, Philippe and Dawson, Jeff},
	month = jul,
	year = {2007},
	pages = {315--317},
}

@article{santos_modulation_2007,
	title = {Modulation of {Frankia} alni {ACN14a} oxidative stress response: activity, expression and phylogeny of catalases},
	volume = {130},
	issn = {0031-9317, 1399-3054},
	shorttitle = {Modulation of {Frankia} alni {ACN14a} oxidative stress response},
	url = {http://doi.wiley.com/10.1111/j.1399-3054.2007.00868.x},
	doi = {10/cvrx56},
	language = {en},
	number = {3},
	urldate = {2021-06-10},
	journal = {Physiologia Plantarum},
	author = {Santos, Catarina L. and Vieira, João and Sellstedt, Anita and Normand, Philippe and Moradas-Ferreira, Pedro and Tavares, Fernando},
	month = jul,
	year = {2007},
	pages = {454--463},
}

@article{tavares_reactive_2007,
	title = {Reactive oxygen species in legume and actinorhizal nitrogen-fixing symbioses: the microsymbiont?s responses to an unfriendly reception},
	volume = {130},
	issn = {0031-9317, 1399-3054},
	shorttitle = {Reactive oxygen species in legume and actinorhizal nitrogen-fixing symbioses},
	url = {http://doi.wiley.com/10.1111/j.1399-3054.2007.00933.x},
	doi = {10/bfbmkh},
	language = {en},
	number = {3},
	urldate = {2021-06-10},
	journal = {Physiologia Plantarum},
	author = {Tavares, Fernando and Santos, Catarina L. and Sellstedt, Anita},
	month = jul,
	year = {2007},
	pages = {344--356},
}

@article{leul_organization_2007,
	title = {The organization, regulation and phylogeny of uptake hydrogenase genes in {Frankia}},
	volume = {130},
	issn = {0031-9317, 1399-3054},
	url = {http://doi.wiley.com/10.1111/j.1399-3054.2007.00861.x},
	doi = {10/fm8kd2},
	language = {en},
	number = {3},
	urldate = {2021-06-10},
	journal = {Physiologia Plantarum},
	author = {Leul, Melakeselam and Normand, Philippe and Sellstedt, Anita},
	month = jul,
	year = {2007},
	pages = {464--470},
}

@article{normand_genome_2006,
	title = {Genome characteristics of facultatively symbiotic {Frankia} sp. strains reflect host range and host plant biogeography},
	volume = {17},
	issn = {1088-9051},
	url = {http://www.genome.org/cgi/doi/10.1101/gr.5798407},
	doi = {10/bjt4n7},
	language = {en},
	number = {1},
	urldate = {2021-06-10},
	journal = {Genome Research},
	author = {Normand, P. and Lapierre, P. and Tisa, L. S. and Gogarten, J. P. and Alloisio, N. and Bagnarol, E. and Bassi, C. A. and Berry, A. M. and Bickhart, D. M. and Choisne, N. and Couloux, A. and Cournoyer, B. and Cruveiller, S. and Daubin, V. and Demange, N. and Francino, M. P. and Goltsman, E. and Huang, Y. and Kopp, O. R. and Labarre, L. and Lapidus, A. and Lavire, C. and Marechal, J. and Martinez, M. and Mastronunzio, J. E. and Mullin, B. C. and Niemann, J. and Pujic, P. and Rawnsley, T. and Rouy, Z. and Schenowitz, C. and Sellstedt, A. and Tavares, F. and Tomkins, J. P. and Vallenet, D. and Valverde, C. and Wall, L. G. and Wang, Y. and Medigue, C. and Benson, D. R.},
	month = dec,
	year = {2006},
	pages = {7--15},
}

@article{mohapatra_occurrence_2006,
	series = {{IHEC} 2005 and {COST} {Action} 841 {Final} {Meeting}},
	title = {Occurrence and characterisation of the hydrogen-evolving enzyme in {Frankia} sp.},
	volume = {31},
	issn = {0360-3199},
	url = {https://www.sciencedirect.com/science/article/pii/S036031990600214X},
	doi = {10.1016/j.ijhydene.2006.06.009},
	abstract = {An increase in hydrogen evolution from the hydrogen-evolving enzyme in the actinomycete Frankia was recorded in the presence of nickel. Immunogold localisation analysis of the intracellular distribution of hydrogenase proteins indicated that they were evenly distributed in the membranes and cytosol of both hyphae and vesicles. In addition, molecular characterisation of the hydrogen-evolving enzyme at the proteomic level, using two-dimensional gel electrophoresis combined with mass spectrometry, confirmed that the Frankia hydrogen-evolving enzyme is similar to the cyanobacterial bidirectional hydrogenase of Anabena siamensis.},
	language = {en},
	number = {11},
	urldate = {2021-06-11},
	journal = {International Journal of Hydrogen Energy},
	author = {Mohapatra, A. and Leul, M. and Sandström, G. and Sellstedt, A.},
	month = sep,
	year = {2006},
	keywords = {Frankia, Hydrogen, Hydrogen-evolving hydrogenase, Nickel, bidirectional hydrogenase, classification, cloning, expression, genes, hydrogen, hydrogen-evolving hydrogenase, kb5, localization, nickel, nife hydrogenase, rhodococcus-opacus},
	pages = {1445--1451},
}

An increase in hydrogen evolution from the hydrogen-evolving enzyme in the actinomycete Frankia was recorded in the presence of nickel. Immunogold localisation analysis of the intracellular distribution of hydrogenase proteins indicated that they were evenly distributed in the membranes and cytosol of both hyphae and vesicles. In addition, molecular characterisation of the hydrogen-evolving enzyme at the proteomic level, using two-dimensional gel electrophoresis combined with mass spectrometry, confirmed that the Frankia hydrogen-evolving enzyme is similar to the cyanobacterial bidirectional hydrogenase of Anabena siamensis.

@article{leul_biodiversity_2005,
	title = {Biodiversity of {Hydrogenases} in {Frankia}},
	volume = {50},
	issn = {1432-0991},
	url = {https://doi.org/10.1007/s00284-004-4323-6},
	doi = {10.1007/s00284-004-4323-6},
	abstract = {Eighteen Frankia strains originally isolated from nine different host plants were used to study the biodiversity of hydrogenase in Frankia. In the physiological analysis, the activities of uptake hydrogenase and bidirectional hydrogenase were performed by monitoring the oxidation of hydrogen after supplying the cells with 1\% hydrogen and the evolution of hydrogen using methyl viologen as an electron donor, respectively. These analyses were supported with a study of the immunological relationship between Frankia hydrogenase and other different known hydrogenases from other microorganisms. Uptake hydrogenase activity was recorded from all the Frankia strains investigated. A methyl-viologen-mediated hydrogen evolution was recorded from only four Frankia strains irrespective of the source of Frankia. From the immunological and physiological studies, we here report that there are at least three types of hydrogenases in Frankia: Ni-Fe uptake hydrogenase, hydrogen-evolving hydrogenase, and [Fe]-hydrogenase. An immunogold localization study, by cryosection technique, of the effect of nickel on the intercellular distribution of hydrogenase proteins in Frankia indicated that nickel affects the transfer of hydrogenase proteins into the membrane.},
	language = {en},
	number = {1},
	urldate = {2021-06-11},
	journal = {Current Microbiology},
	author = {Leul, Melakeselam and Mohapatra, Anasuya and Sellstedt, Anita},
	month = jan,
	year = {2005},
	pages = {17--23},
}

Eighteen Frankia strains originally isolated from nine different host plants were used to study the biodiversity of hydrogenase in Frankia. In the physiological analysis, the activities of uptake hydrogenase and bidirectional hydrogenase were performed by monitoring the oxidation of hydrogen after supplying the cells with 1% hydrogen and the evolution of hydrogen using methyl viologen as an electron donor, respectively. These analyses were supported with a study of the immunological relationship between Frankia hydrogenase and other different known hydrogenases from other microorganisms. Uptake hydrogenase activity was recorded from all the Frankia strains investigated. A methyl-viologen-mediated hydrogen evolution was recorded from only four Frankia strains irrespective of the source of Frankia. From the immunological and physiological studies, we here report that there are at least three types of hydrogenases in Frankia: Ni-Fe uptake hydrogenase, hydrogen-evolving hydrogenase, and [Fe]-hydrogenase. An immunogold localization study, by cryosection technique, of the effect of nickel on the intercellular distribution of hydrogenase proteins in Frankia indicated that nickel affects the transfer of hydrogenase proteins into the membrane.

@article{stahl_measuring_2005,
	title = {Measuring nitrogen fixation by {Sesbania} sesban planted fallows using {15N} tracer technique in {Kenya}},
	volume = {65},
	issn = {1572-9680},
	url = {https://doi.org/10.1007/s10457-004-6072-8},
	doi = {10.1007/s10457-004-6072-8},
	abstract = {A field experiment was performed in eastern Kenya to estimate N2 fixation by Sesbania sesban over an 18-month period using the 15N dilution method. The influence of three reference species, Senna spectabilis, Eucalyptus saligna and Grevillea robusta, on the estimates of N2 fixation was also assessed. Percentage Ndfa (nitrogen derived from the atmosphere) was calculated based on foliar atom excess (FAE), above-ground atom excess (AAE) or whole tree atom excess (WAE) data. The differences in atom\% 15N excess values between species and plant parts are presented and discussed. We recommend the use of several reference species for estimating \%Ndfa and that the different results obtained should be carefully considered in relation to the issues being addressed. In this study, Senna was the most suitable of the three reference species because its N uptake pattern and phenology were very similar to those of Sesbania. When well established, the amount of N fixed by Sesbania accounts for more than 80\% of its total N content, according to FAE-based estimates. We estimated the Ndfa by Sesbania after 18 months to between 500 and 600 kg ha−1 , depending on whether FAE, AAE or WAE data were used and on the choice of reference species. The substantial accumulation of N in planted Sesbania highlighted its potential to increase the sustainability of crop production on N-limited soils. We consider the 15N dilution method to be appropriate for quantifying N2 fixation in improved fallows in studies, similar to this one, of young trees with high N2-fixing ability.},
	language = {en},
	number = {1},
	urldate = {2021-06-11},
	journal = {Agroforestry Systems},
	author = {Ståhl, Lena and Högberg, Peter and Sellstedt, Anita and Buresh, Roland J.},
	month = oct,
	year = {2005},
	pages = {67--79},
}

A field experiment was performed in eastern Kenya to estimate N2 fixation by Sesbania sesban over an 18-month period using the 15N dilution method. The influence of three reference species, Senna spectabilis, Eucalyptus saligna and Grevillea robusta, on the estimates of N2 fixation was also assessed. Percentage Ndfa (nitrogen derived from the atmosphere) was calculated based on foliar atom excess (FAE), above-ground atom excess (AAE) or whole tree atom excess (WAE) data. The differences in atom% 15N excess values between species and plant parts are presented and discussed. We recommend the use of several reference species for estimating %Ndfa and that the different results obtained should be carefully considered in relation to the issues being addressed. In this study, Senna was the most suitable of the three reference species because its N uptake pattern and phenology were very similar to those of Sesbania. When well established, the amount of N fixed by Sesbania accounts for more than 80% of its total N content, according to FAE-based estimates. We estimated the Ndfa by Sesbania after 18 months to between 500 and 600 kg ha−1 , depending on whether FAE, AAE or WAE data were used and on the choice of reference species. The substantial accumulation of N in planted Sesbania highlighted its potential to increase the sustainability of crop production on N-limited soils. We consider the 15N dilution method to be appropriate for quantifying N2 fixation in improved fallows in studies, similar to this one, of young trees with high N2-fixing ability.

@article{leul_molecular_2005,
	title = {Molecular characterization of uptake hydrogenase in {Frankia}},
	volume = {33},
	issn = {0300-5127},
	url = {https://doi.org/10.1042/BST0330064},
	doi = {10.1042/BST0330064},
	abstract = {A molecular characterization of uptake hydrogenase in Frankia was performed by using two-dimensional gel electrophoresis, matrix-assisted laser-desorption ionization–time-of-flight mass spectrometry, PCR amplification and Southern blotting. A polypeptide of approx. 60 kDa was recognized in Frankia UGL011102, AVCI1 and KB5 on the two-dimensional gel by blotting with Ralstonia eutropha (Hox G) antibody. Further analysis by MS resulted in a peptide ‘fingerprint’, which was similar to the membrane-bound hydrogenase 2 large subunit (HYD2) in Escherichia coli. In addition, a 127 bp PCR fragment could also be amplified from Frankia AVCI1, which gave a 76\% similarity with the large subunit of hydrogenase in, e.g., Azotobacter chrococcum, Bradyrhizobium japonicum and Rhizobium leguminosarum. Although immunological similarity between the small subunit of Frankia hydrogenase and that of other organisms has not yet been found, a PCR product of 500 bp could be amplified from the local source of Frankia, the analysis of which gave 69 and 67\% identity with the small subunit of hydrogenases in B. japonicum and R. leguminosarum respectively. A Southern-blot analysis further indicated evidence for the presence of the small hydrogenase subunit in other Frankia strains, i.e. KB5, AvcI1 and CcI3.},
	number = {1},
	urldate = {2021-06-11},
	journal = {Biochemical Society Transactions},
	author = {Leul, M. and Mattsson, U. and Sellstedt, A.},
	month = feb,
	year = {2005},
	pages = {64--66},
}

A molecular characterization of uptake hydrogenase in Frankia was performed by using two-dimensional gel electrophoresis, matrix-assisted laser-desorption ionization–time-of-flight mass spectrometry, PCR amplification and Southern blotting. A polypeptide of approx. 60 kDa was recognized in Frankia UGL011102, AVCI1 and KB5 on the two-dimensional gel by blotting with Ralstonia eutropha (Hox G) antibody. Further analysis by MS resulted in a peptide ‘fingerprint’, which was similar to the membrane-bound hydrogenase 2 large subunit (HYD2) in Escherichia coli. In addition, a 127 bp PCR fragment could also be amplified from Frankia AVCI1, which gave a 76% similarity with the large subunit of hydrogenase in, e.g., Azotobacter chrococcum, Bradyrhizobium japonicum and Rhizobium leguminosarum. Although immunological similarity between the small subunit of Frankia hydrogenase and that of other organisms has not yet been found, a PCR product of 500 bp could be amplified from the local source of Frankia, the analysis of which gave 69 and 67% identity with the small subunit of hydrogenases in B. japonicum and R. leguminosarum respectively. A Southern-blot analysis further indicated evidence for the presence of the small hydrogenase subunit in other Frankia strains, i.e. KB5, AvcI1 and CcI3.

@article{gentili_physiological_2005,
	title = {Physiological and molecular diversity of feather moss associative {N}-2-fixing cyanobacteria},
	volume = {56},
	issn = {0022-0957},
	doi = {10.1093/jxb/eri309},
	abstract = {Cyanobacteria colonizing the feather moss Pleurozium schreberi were isolated from moss samples collected in northern Sweden and subjected to physiological and molecular characterization. Morphological studies of isolated and moss-associated cyanobacteria were carried out by light microscopy. Molecular tools were used for cyanobacteria identification, and a reconstitution experiment of the association between non-associative mosses and cyanobacteria was conducted. The influence of temperature on N-2 fixation in the different cyanobacterial isolates and the influence of light and temperature on N-2-fixation rates in the moss were studied using the acetylene reduction assay. Two different cyanobacteria were effectively isolated from P. schreberi: Nostoc sp. and Calothrix sp. A third genus, Stigonema sp. was identified by microscopy, but could not be isolated. The Nostoc sp. was found to fix N-2 at lower temperatures than Calothrix sp. Nostoc sp. and Stigonema sp. were the predominant cyanobacteria colonizing the moss. The attempt to reconstitute the association between the moss and cyanobacteria was successful. The two isolated genera of cyanobacteria in feather moss samples collected in northern Sweden differ in their temperature optima, which may have important ecological implications.},
	language = {English},
	number = {422},
	journal = {Journal of Experimental Botany},
	author = {Gentili, F. and Nilsson, M. C. and Zackrisson, O. and DeLuca, T. H. and Sellstedt, A.},
	month = dec,
	year = {2005},
	note = {Place: Oxford
Publisher: Oxford Univ Press
WOS:000233491300011},
	keywords = {Calothrix, N-2   fixation Nostoc, Stigonema, abiotic factors, acetylene reduction assay (ARA), antarctica, biological nitrogen-fixation, boreal forests, communities, cyanobacteria, devon-island, ecosystems, lowland, moss, svalbard, vegetation},
	pages = {3121--3127},
}

Cyanobacteria colonizing the feather moss Pleurozium schreberi were isolated from moss samples collected in northern Sweden and subjected to physiological and molecular characterization. Morphological studies of isolated and moss-associated cyanobacteria were carried out by light microscopy. Molecular tools were used for cyanobacteria identification, and a reconstitution experiment of the association between non-associative mosses and cyanobacteria was conducted. The influence of temperature on N-2 fixation in the different cyanobacterial isolates and the influence of light and temperature on N-2-fixation rates in the moss were studied using the acetylene reduction assay. Two different cyanobacteria were effectively isolated from P. schreberi: Nostoc sp. and Calothrix sp. A third genus, Stigonema sp. was identified by microscopy, but could not be isolated. The Nostoc sp. was found to fix N-2 at lower temperatures than Calothrix sp. Nostoc sp. and Stigonema sp. were the predominant cyanobacteria colonizing the moss. The attempt to reconstitute the association between the moss and cyanobacteria was successful. The two isolated genera of cyanobacteria in feather moss samples collected in northern Sweden differ in their temperature optima, which may have important ecological implications.

@article{mohapatra_hydrogen-evolving_2004,
	title = {A hydrogen-evolving enzyme is present in {Frankia} sp. {R43}},
	volume = {236},
	issn = {0378-1097},
	url = {https://doi.org/10.1111/j.1574-6968.2004.tb09652.x},
	doi = {10.1111/j.1574-6968.2004.tb09652.x},
	abstract = {The ability to evolve hydrogen using methyl viologen as an electron donor was assayed in the nitrogen-fixing actinomycetes Frankia sp. R43 and Frankia sp. KB5. To further examine the nature of hydrogen-evolving enzymes that may be present in these organisms immunological studies were performed. Under anaerobic conditions (both nitrogen-limiting and nitrogen-containing) Frankia sp. R43 but not Frankia sp. KB5 evolved hydrogen, which was not linked to NAD-reducing activity. Immunological analysis of total protein from Frankia sp. R43 and Frankia sp. KB5 using an antiserum raised against Ralstonia eutropha HoxF, recognized an antigen in Frankia sp. R43 but not in Frankia sp. KB5. Immunogold labeling using antibodies raised against the R. eutropha HoxH recognized sites in both hyphae and vesicles of Frankia sp. R43, but not in Frankia sp. KB5. Based on these physiological and immunological findings, we conclude that Frankia sp. R43 has a hydrogen-evolving hydrogenase.},
	number = {2},
	urldate = {2021-06-30},
	journal = {FEMS Microbiology Letters},
	author = {Mohapatra, Anasuya and Leul, Melakeselam and Mattsson, Ulrika and Sellstedt, Anita},
	month = jul,
	year = {2004},
	pages = {235--240},
}

The ability to evolve hydrogen using methyl viologen as an electron donor was assayed in the nitrogen-fixing actinomycetes Frankia sp. R43 and Frankia sp. KB5. To further examine the nature of hydrogen-evolving enzymes that may be present in these organisms immunological studies were performed. Under anaerobic conditions (both nitrogen-limiting and nitrogen-containing) Frankia sp. R43 but not Frankia sp. KB5 evolved hydrogen, which was not linked to NAD-reducing activity. Immunological analysis of total protein from Frankia sp. R43 and Frankia sp. KB5 using an antiserum raised against Ralstonia eutropha HoxF, recognized an antigen in Frankia sp. R43 but not in Frankia sp. KB5. Immunogold labeling using antibodies raised against the R. eutropha HoxH recognized sites in both hyphae and vesicles of Frankia sp. R43, but not in Frankia sp. KB5. Based on these physiological and immunological findings, we conclude that Frankia sp. R43 has a hydrogen-evolving hydrogenase.

@article{zackrisson_nitrogen_2004,
	title = {Nitrogen fixation increases with successional age in boreal forests},
	volume = {85},
	issn = {0012-9658},
	doi = {10.1890/04-0461},
	abstract = {There is little understanding of successional dynamics of N fixation in northern boreal forests. Recent evidence suggests that N fixation by cyanobacteria in association with the common feather moss Pleurozium schreberi contributes to a significant proportion of the total N economy. The Purpose Of the work herein was to determine how time since last fire influences N fixation rates in boreal forests. We evaluated seasonal N fixation rates oil a total of 12 natural forest preserves varying in time since last fire (35-355 years). Each site was monitored for N fixation activity using a calibrated acetylene reduction assay. Nitrogen fixation rates were found to increase linearly with time since fire. This increase in N fixation with succession is likely a function of degree of colonization by cyanobacteria and site factors Such as presence of available N. Surface applications of 4.5 kg N(.)ha (1.)yr(-1) as NH4NO3 Were found to eliminate N fixation while applications of P resulted in only a slight and temporary increase of N fixation rates. In contrast to common observation our findings suggest that N fixation in boreal forests becomes more important in late Succession. Limited N availability in late Succession is clearly one of the primary drivers of N fixation rates in boreal forest ecosystems. These findings may help to explain the origin of high rates of net N accumulation in Soil unaccounted for at northern boreal sites.},
	language = {English},
	number = {12},
	journal = {Ecology},
	author = {Zackrisson, O. and DeLuca, T. H. and Nilsson, M. C. and Sellstedt, A. and Berglund, L. M.},
	month = dec,
	year = {2004},
	note = {Place: Hoboken
Publisher: Wiley
WOS:000226297500014},
	keywords = {Pleurozium schreberi, Sweden, abiotic factors, accumulation, acetylene, acetylene reduction, boreal forest, cyanobacteria, ecosystem properties, feather mosses, fire, gradient, hawaii, island area, nitrogen   fixation, photosynthesis, plants, succession},
	pages = {3327--3334},
}

There is little understanding of successional dynamics of N fixation in northern boreal forests. Recent evidence suggests that N fixation by cyanobacteria in association with the common feather moss Pleurozium schreberi contributes to a significant proportion of the total N economy. The Purpose Of the work herein was to determine how time since last fire influences N fixation rates in boreal forests. We evaluated seasonal N fixation rates oil a total of 12 natural forest preserves varying in time since last fire (35-355 years). Each site was monitored for N fixation activity using a calibrated acetylene reduction assay. Nitrogen fixation rates were found to increase linearly with time since fire. This increase in N fixation with succession is likely a function of degree of colonization by cyanobacteria and site factors Such as presence of available N. Surface applications of 4.5 kg N(.)ha (1.)yr(-1) as NH4NO3 Were found to eliminate N fixation while applications of P resulted in only a slight and temporary increase of N fixation rates. In contrast to common observation our findings suggest that N fixation in boreal forests becomes more important in late Succession. Limited N availability in late Succession is clearly one of the primary drivers of N fixation rates in boreal forest ecosystems. These findings may help to explain the origin of high rates of net N accumulation in Soil unaccounted for at northern boreal sites.

@article{leitz_laser-based_2003,
	title = {Laser-based micromanipulation for separation and identification of individual {Frankia} vesicles},
	volume = {224},
	issn = {0378-1097},
	doi = {10/bf78w8},
	abstract = {In studies of symbiotic efficiency it is of great importance to identify and separate individual Frankia strains from a nodule. Therefore, a new laser-based micromanipulation technique has been developed in which individual vesicles from root nodules of two Frankia-Alnus symbioses have been successfully cut loose and separated from clusters of vesicles in sterile conditions under light microscopy using a laser scalpel and optical tweezers. Vesicles from the Alnus incana-Frankia AvCIl symbiosis were successfully isolated and grown in culture using this technique. The DNA from both Frankia sources was amplified by polymerase chain reaction (PCR). The work shows that a combination of laser-based manipulation techniques and PCR can be used for the separation and study of individual vesicles. This novel laser-based micromanipulation technique opens up various new possibilities, for instance, to study whether several Frankia strains can grow simultaneously in the same root nodule. (C) 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.},
	language = {English},
	number = {1},
	journal = {Fems Microbiology Letters},
	author = {Leitz, G. and Lundberg, C. and Fallman, E. and Axner, O. and Sellstedt, A.},
	month = jul,
	year = {2003},
	note = {Place: Amsterdam
Publisher: Elsevier Science Bv
WOS:000184248400014},
	keywords = {Frankia, bacteria, casuarina, dna, hydrogen metabolism, laser scalpel, manipulation, nitrogen-fixation, optical tweezers, pcr, polymerase chain-reaction, rflp, root-nodules, strains},
	pages = {97--100},
}

In studies of symbiotic efficiency it is of great importance to identify and separate individual Frankia strains from a nodule. Therefore, a new laser-based micromanipulation technique has been developed in which individual vesicles from root nodules of two Frankia-Alnus symbioses have been successfully cut loose and separated from clusters of vesicles in sterile conditions under light microscopy using a laser scalpel and optical tweezers. Vesicles from the Alnus incana-Frankia AvCIl symbiosis were successfully isolated and grown in culture using this technique. The DNA from both Frankia sources was amplified by polymerase chain reaction (PCR). The work shows that a combination of laser-based manipulation techniques and PCR can be used for the separation and study of individual vesicles. This novel laser-based micromanipulation technique opens up various new possibilities, for instance, to study whether several Frankia strains can grow simultaneously in the same root nodule. (C) 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

@article{mattsson_nickel_2002,
	title = {Nickel {Affects} {Activity} {More} {Than} {Expression} of {Hydrogenase} {Protein} in {Frankia}},
	volume = {44},
	issn = {1432-0991},
	url = {https://doi.org/10.1007/s00284-001-0056-y},
	doi = {10.1007/s00284-001-0056-y},
	language = {en},
	number = {2},
	urldate = {2021-10-19},
	journal = {Current Microbiology},
	author = {Mattsson, Ulrika and Sellstedt, Anita},
	month = feb,
	year = {2002},
	pages = {88--93},
}

@article{deluca_quantifying_2002,
	title = {Quantifying nitrogen-fixation in feather moss carpets of boreal forests},
	volume = {419},
	copyright = {2002 Macmillan Magazines Ltd.},
	issn = {1476-4687},
	url = {https://www.nature.com/articles/nature01051},
	doi = {10/bp7gfx},
	abstract = {Biological nitrogen (N) fixation is the primary source of N within natural ecosystems1, yet the origin of boreal forest N has remained elusive. The boreal forests of Eurasia and North America lack any significant, widespread symbiotic N-fixing plants1,2,3,4,5,6. With the exception of scattered stands of alder in early primary successional forests7, N-fixation in boreal forests is considered to be extremely limited. Nitrogen-fixation in northern European boreal forests has been estimated2 at only 0.5 kg N ha-1 yr-1; however, organic N is accumulated in these ecosystems at a rate of 3 kg N ha-1 yr-1 (ref. 8). Our limited understanding of the origin of boreal N is unacceptable given the extent of the boreal forest region, but predictable given our imperfect knowledge of N-fixation1,9. Herein we report on a N-fixing symbiosis between a cyanobacterium (Nostoc sp.) and the ubiquitous feather moss, Pleurozium schreberi (Bird) Mitt. that alone fixes between 1.5 and 2.0 kg N ha-1 yr-1 in mid- to late-successional forests of northern Scandinavia and Finland. Previous efforts have probably underestimated N-fixation potential in boreal forests.},
	language = {en},
	number = {6910},
	urldate = {2021-10-19},
	journal = {Nature},
	author = {DeLuca, Thomas H. and Zackrisson, Olle and Nilsson, Marie-Charlotte and Sellstedt, Anita},
	month = oct,
	year = {2002},
	note = {Bandiera\_abtest: a
Cg\_type: Nature Research Journals
Number: 6910
Primary\_atype: Research
Publisher: Nature Publishing Group},
	pages = {917--920},
}

Biological nitrogen (N) fixation is the primary source of N within natural ecosystems1, yet the origin of boreal forest N has remained elusive. The boreal forests of Eurasia and North America lack any significant, widespread symbiotic N-fixing plants1,2,3,4,5,6. With the exception of scattered stands of alder in early primary successional forests7, N-fixation in boreal forests is considered to be extremely limited. Nitrogen-fixation in northern European boreal forests has been estimated2 at only 0.5 kg N ha-1 yr-1; however, organic N is accumulated in these ecosystems at a rate of 3 kg N ha-1 yr-1 (ref. 8). Our limited understanding of the origin of boreal N is unacceptable given the extent of the boreal forest region, but predictable given our imperfect knowledge of N-fixation1,9. Herein we report on a N-fixing symbiosis between a cyanobacterium (Nostoc sp.) and the ubiquitous feather moss, Pleurozium schreberi (Bird) Mitt. that alone fixes between 1.5 and 2.0 kg N ha-1 yr-1 in mid- to late-successional forests of northern Scandinavia and Finland. Previous efforts have probably underestimated N-fixation potential in boreal forests.

@article{tavares_dnase-resistant_2001,
	title = {{DNase}-{Resistant} {DNA} in the {Extracellular} and {Cell} {Wall}-{Associated} {Fractions} of {Frankia} {Strains} {R43} and {CcI3}},
	volume = {42},
	issn = {1432-0991},
	url = {https://doi.org/10.1007/s002840010198},
	doi = {10/dgzrnw},
	abstract = {DNases were shown to be present in the extracellular fraction of Frankia strains R43 and CcI3. In spite of this, DNA was found in both the extracellular and cell wall fractions of these strains, and it was shown that extracellular DNA was resistant to the DNases secreted into the culture medium of both Frankia strains. Furthermore, Southern blot analysis under high stringency conditions revealed the chromosomal origin of the cell wall-adsorbed DNA (CW-DNA). Mobility gel band shift assays suggested that the extracellular DNA and the CW-DNA are engaged in complexes with other molecules, most likely proteins, which are probably responsible for the enzymatic resistance observed against extracellular DNase activities. In addition, it was shown that lysis of a small proportion of the cells in the exponential growth phase may account for the DNA being released into the supernatant and adsorbed to the cell wall.},
	language = {en},
	number = {3},
	urldate = {2021-11-02},
	journal = {Current Microbiology},
	author = {Tavares, Fernando and Sellstedt, Anita},
	month = mar,
	year = {2001},
	pages = {168--172},
}

DNases were shown to be present in the extracellular fraction of Frankia strains R43 and CcI3. In spite of this, DNA was found in both the extracellular and cell wall fractions of these strains, and it was shown that extracellular DNA was resistant to the DNases secreted into the culture medium of both Frankia strains. Furthermore, Southern blot analysis under high stringency conditions revealed the chromosomal origin of the cell wall-adsorbed DNA (CW-DNA). Mobility gel band shift assays suggested that the extracellular DNA and the CW-DNA are engaged in complexes with other molecules, most likely proteins, which are probably responsible for the enzymatic resistance observed against extracellular DNase activities. In addition, it was shown that lysis of a small proportion of the cells in the exponential growth phase may account for the DNA being released into the supernatant and adsorbed to the cell wall.

@article{mattsson_frankia_2001,
	title = {Frankia {KB5} {Possesses} a {Hydrogenase} {Immunologically} {Related} to {Membrane}-{Bound} [{NiFe}]-{Hydrogenases}},
	volume = {42},
	issn = {1432-0991},
	url = {https://doi.org/10.1007/s002840010244},
	doi = {10.1007/s002840010244},
	abstract = {The immunological relationship of the hydrogenase in Frankia KB5 to hydrogenases in other microorganisms was investigated using antisera raised against holo-[NiFe]-hydrogenases isolated from Alcaligenes latus, Azotobacter vinelandii, Ralstonia eutropha, and the small and large hydrogenase subunits from Bradyrhizobium japonicum. The antisera raised against the A. latus, R. eutropha, and B. japonicum (large subunit) polypeptides were found to recognize two polypeptides, corresponding to the unprocessed and processed forms of the hydrogenase subunit in Frankia KB5. None of the antisera, including the antibodies produced against the small hydrogenase subunit isolated from B. japonicum, recognized any polypeptide related to the small hydrogenase subunit in Frankia KB5. An immunogold localization study of the intracellular distribution of hydrogenase in Frankia KB5, with the cryo-section technique, showed that labeling in the membrane of both hyphae and vesicles was positively correlated with hydrogenase activity.},
	language = {en},
	number = {6},
	urldate = {2021-11-02},
	journal = {Current Microbiology},
	author = {Mattsson, Ulrika and Johansson, Lenore and Sandström, Gunnar and Sellstedt, Anita},
	month = jun,
	year = {2001},
	pages = {438--441},
}

The immunological relationship of the hydrogenase in Frankia KB5 to hydrogenases in other microorganisms was investigated using antisera raised against holo-[NiFe]-hydrogenases isolated from Alcaligenes latus, Azotobacter vinelandii, Ralstonia eutropha, and the small and large hydrogenase subunits from Bradyrhizobium japonicum. The antisera raised against the A. latus, R. eutropha, and B. japonicum (large subunit) polypeptides were found to recognize two polypeptides, corresponding to the unprocessed and processed forms of the hydrogenase subunit in Frankia KB5. None of the antisera, including the antibodies produced against the small hydrogenase subunit isolated from B. japonicum, recognized any polypeptide related to the small hydrogenase subunit in Frankia KB5. An immunogold localization study of the intracellular distribution of hydrogenase in Frankia KB5, with the cryo-section technique, showed that labeling in the membrane of both hyphae and vesicles was positively correlated with hydrogenase activity.

@article{tavares_simple_2000,
	title = {A simple, rapid and non-destructive procedure to extract cell wall-associated proteins from {Frankia}},
	volume = {39},
	issn = {0167-7012},
	url = {https://www.sciencedirect.com/science/article/pii/S0167701299001153},
	doi = {10.1016/S0167-7012(99)00115-3},
	abstract = {A simple cell fractionation procedure was developed to extract cell wall-associated proteins from the nitrogen-fixing actinomycete Frankia. The method was based on washing Frankia mycelia in 62.5 mM Tris–HCl (pH 6.8) buffer supplemented with 0.1\% Triton X-100 as solubilizing agent. Cell wall-associated proteins were efficiently extracted in less than 10 min, recovering approximately 94.5±7.44 μg protein per extraction procedure from exponentially growing cells corresponding to 50 ml of culture. The amount of cell lysis occurring during the cell wall extraction was estimated to be 1.50±0.51\%. Three peptidoglycan hydrolases with apparent molecular masses of 4.7, 12.1, and 17.8 kDa were detected by zymography in the cell wall-associated protein fraction. On the contrary, no cell wall lytic enzyme was detected in the cytoplasmic protein fraction. These results indicate that the present method enables a specific extraction of cell wall-associated proteins. Moreover, fluorescein isothiocyanate (FITC) labelling of the cell surface proteins showed an efficient removal of cell wall-associated proteins. Growth of the treated Frankia cells (i.e. cells from which the cell wall-associated proteins were removed) in semi-solid media suggested that these cells were still viable. This technique is of importance for functionality studies of cell wall-associated proteins, particularly for bacteria where traditional cell fractionation methods are difficult to be applied.},
	language = {en},
	number = {2},
	urldate = {2021-11-08},
	journal = {Journal of Microbiological Methods},
	author = {Tavares, Fernando and Sellstedt, Anita},
	month = jan,
	year = {2000},
	keywords = {Actinomycetes, Cell fractionation, Gram-positive cell wall, Peptidoglycan hydrolases},
	pages = {171--178},
}

A simple cell fractionation procedure was developed to extract cell wall-associated proteins from the nitrogen-fixing actinomycete Frankia. The method was based on washing Frankia mycelia in 62.5 mM Tris–HCl (pH 6.8) buffer supplemented with 0.1% Triton X-100 as solubilizing agent. Cell wall-associated proteins were efficiently extracted in less than 10 min, recovering approximately 94.5±7.44 μg protein per extraction procedure from exponentially growing cells corresponding to 50 ml of culture. The amount of cell lysis occurring during the cell wall extraction was estimated to be 1.50±0.51%. Three peptidoglycan hydrolases with apparent molecular masses of 4.7, 12.1, and 17.8 kDa were detected by zymography in the cell wall-associated protein fraction. On the contrary, no cell wall lytic enzyme was detected in the cytoplasmic protein fraction. These results indicate that the present method enables a specific extraction of cell wall-associated proteins. Moreover, fluorescein isothiocyanate (FITC) labelling of the cell surface proteins showed an efficient removal of cell wall-associated proteins. Growth of the treated Frankia cells (i.e. cells from which the cell wall-associated proteins were removed) in semi-solid media suggested that these cells were still viable. This technique is of importance for functionality studies of cell wall-associated proteins, particularly for bacteria where traditional cell fractionation methods are difficult to be applied.

@article{mattsson_hydrogenase_2000,
	title = {Hydrogenase in {Frankia} {KB5}: {Expression} of and relation to nitrogenase},
	volume = {46},
	issn = {0008-4166},
	shorttitle = {Hydrogenase in {Frankia} {KB5}},
	url = {https://cdnsciencepub.com/doi/10.1139/w00-100},
	doi = {10.1139/w00-100},
	number = {12},
	urldate = {2021-11-08},
	journal = {Canadian Journal of Microbiology},
	author = {Mattsson, Ulrika and Sellstedt, Anita},
	month = dec,
	year = {2000},
	note = {Publisher: NRC Research Press},
	pages = {1091--1095},
}