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Papers by Nicholas Bennette

Research paper thumbnail of Identification and quantification of water-soluble metabolites by cryoprobe-assisted nuclear magnetic resonance spectroscopy applied to microbial fermentation

Magnetic Resonance in Chemistry, 2009

We highlight a range of cryoprobe-assisted NMR methods for studying metabolite production by cyan... more We highlight a range of cryoprobe-assisted NMR methods for studying metabolite production by cyanobacteria, which should be valuable for a wide range of biological applications requiring ultrasensitivity and precise concentration determination over a large dynamic range. Cyroprobe-assisted 1H and 13C NMR have been applied to precise determination of metabolic products excreted during autofermentation in two cyanobacterial species: filamentous Arthrospira (Spirulina) maxima CS-328 and unicellular Synechococcus sp. PCC 7002. Several fermentative end products were identified and quantified in concentrations ranging from 50 to 3000 µM in cell-free media (a direct measurement of native-like samples) with less than 5.5% relative error in under 10 min of acquisition per sample with the assistance of an efficient water-suppression protocol. Relaxation times (T1) of these metabolites in aqueous (1H2O) solution were measured and found to vary by nearly threefold, necessitating generation of individual calibration curves for each species for highest precision. However, using a 4.5 × longer overall recycle delay between scans, the metabolite concentrations can be predicted within 25% error by calibrating only to a single calibration standard (succinate); other metabolites are then calculated on the basis of their signal integrals and known proton degeneracies. Precise ratios of concentrations of 13C-labeled versus unlabeled metabolites were determined from integral ratios of 1H peaks that exhibit 13C1H J-couplings and independently confirmed by direct measurement of areas of corresponding 13C resonances. 13C NMR was used to identify and quantify production of osmolytes, trehalose, and glucosylglycerol by A. maxima. Copyright © 2009 John Wiley & Sons, Ltd.

Research paper thumbnail of Aquatic phototrophs: efficient alternatives to land-based crops for biofuels

Current Opinion in Biotechnology, 2008

To mitigate some of the potentially deleterious environmental and agricultural consequences assoc... more To mitigate some of the potentially deleterious environmental and agricultural consequences associated with current land-based-biofuel feedstocks, we propose the use of biofuels derived from aquatic microbial oxygenic photoautotrophs (AMOPs), more commonly known as cyanobacteria, algae, and diatoms. Herein we review their demonstrated productivity in mass culturing and aspects of their physiology that are particularly attractive for integration into renewable biofuel applications. Compared with terrestrial crops, AMOPs are inherently more efficient solar collectors, use less or no land, can be converted to liquid fuels using simpler technologies than cellulose, and offer secondary uses that fossil fuels do not provide. AMOPs pose a new set of technological challenges if they are to contribute as biofuel feedstocks.

Research paper thumbnail of Redirecting Reductant Flux into Hydrogen Production via Metabolic Engineering of Fermentative Carbon Metabolism in a Cyanobacterium

Applied and Environmental Microbiology, 2010

Some aquatic microbial oxygenic photoautotrophs (AMOPs) make hydrogen (H 2 ), a carbon-neutral, r... more Some aquatic microbial oxygenic photoautotrophs (AMOPs) make hydrogen (H 2 ), a carbon-neutral, renewable product derived from water, in low yields during autofermentation (anaerobic metabolism) of intracellular carbohydrates previously stored during aerobic photosynthesis. We have constructed a mutant (the ldhA mutant) of the cyanobacterium Synechococcus sp. strain PCC 7002 lacking the enzyme for the NADH-dependent reduction of pyruvate to D-lactate, the major fermentative reductant sink in this AMOP. Both nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography-mass spectrometry (LC-MS) metabolomic methods have shown that autofermentation by the ldhA mutant resulted in no D-lactate production and higher concentrations of excreted acetate, alanine, succinate, and hydrogen (up to 5-fold) compared to that by the wild type.

Research paper thumbnail of Identification and quantification of water-soluble metabolites by cryoprobe-assisted nuclear magnetic resonance spectroscopy applied to microbial fermentation

Magnetic Resonance in Chemistry, 2009

We highlight a range of cryoprobe-assisted NMR methods for studying metabolite production by cyan... more We highlight a range of cryoprobe-assisted NMR methods for studying metabolite production by cyanobacteria, which should be valuable for a wide range of biological applications requiring ultrasensitivity and precise concentration determination over a large dynamic range. Cyroprobe-assisted 1 H and 13 C NMR have been applied to precise determination of metabolic products excreted during autofermentation in two cyanobacterial species: filamentous Arthrospira (Spirulina) maxima CS-328 and unicellular Synechococcus sp. PCC 7002. Several fermentative end products were identified and quantified in concentrations ranging from 50 to 3000 µM in cell-free media (a direct measurement of native-like samples) with less than 5.5% relative error in under 10 min of acquisition per sample with the assistance of an efficient water-suppression protocol. Relaxation times (T1) of these metabolites in aqueous ( 1 H 2 O) solution were measured and found to vary by nearly threefold, necessitating generation of individual calibration curves for each species for highest precision. However, using a 4.5 × longer overall recycle delay between scans, the metabolite concentrations can be predicted within 25% error by calibrating only to a single calibration standard (succinate); other metabolites are then calculated on the basis of their signal integrals and known proton degeneracies. Precise ratios of concentrations of 13 C-labeled versus unlabeled metabolites were determined from integral ratios of 1 H peaks that exhibit 13 C-1 H J-couplings and independently confirmed by direct measurement of areas of corresponding 13 C resonances. 13 C NMR was used to identify and quantify production of osmolytes, trehalose, and glucosylglycerol by A. maxima.

Research paper thumbnail of Identification and quantification of water-soluble metabolites by cryoprobe-assisted nuclear magnetic resonance spectroscopy applied to microbial fermentation

Magnetic Resonance in Chemistry, 2009

We highlight a range of cryoprobe-assisted NMR methods for studying metabolite production by cyan... more We highlight a range of cryoprobe-assisted NMR methods for studying metabolite production by cyanobacteria, which should be valuable for a wide range of biological applications requiring ultrasensitivity and precise concentration determination over a large dynamic range. Cyroprobe-assisted 1H and 13C NMR have been applied to precise determination of metabolic products excreted during autofermentation in two cyanobacterial species: filamentous Arthrospira (Spirulina) maxima CS-328 and unicellular Synechococcus sp. PCC 7002. Several fermentative end products were identified and quantified in concentrations ranging from 50 to 3000 µM in cell-free media (a direct measurement of native-like samples) with less than 5.5% relative error in under 10 min of acquisition per sample with the assistance of an efficient water-suppression protocol. Relaxation times (T1) of these metabolites in aqueous (1H2O) solution were measured and found to vary by nearly threefold, necessitating generation of individual calibration curves for each species for highest precision. However, using a 4.5 × longer overall recycle delay between scans, the metabolite concentrations can be predicted within 25% error by calibrating only to a single calibration standard (succinate); other metabolites are then calculated on the basis of their signal integrals and known proton degeneracies. Precise ratios of concentrations of 13C-labeled versus unlabeled metabolites were determined from integral ratios of 1H peaks that exhibit 13C1H J-couplings and independently confirmed by direct measurement of areas of corresponding 13C resonances. 13C NMR was used to identify and quantify production of osmolytes, trehalose, and glucosylglycerol by A. maxima. Copyright © 2009 John Wiley & Sons, Ltd.

Research paper thumbnail of Aquatic phototrophs: efficient alternatives to land-based crops for biofuels

Current Opinion in Biotechnology, 2008

To mitigate some of the potentially deleterious environmental and agricultural consequences assoc... more To mitigate some of the potentially deleterious environmental and agricultural consequences associated with current land-based-biofuel feedstocks, we propose the use of biofuels derived from aquatic microbial oxygenic photoautotrophs (AMOPs), more commonly known as cyanobacteria, algae, and diatoms. Herein we review their demonstrated productivity in mass culturing and aspects of their physiology that are particularly attractive for integration into renewable biofuel applications. Compared with terrestrial crops, AMOPs are inherently more efficient solar collectors, use less or no land, can be converted to liquid fuels using simpler technologies than cellulose, and offer secondary uses that fossil fuels do not provide. AMOPs pose a new set of technological challenges if they are to contribute as biofuel feedstocks.

Research paper thumbnail of Redirecting Reductant Flux into Hydrogen Production via Metabolic Engineering of Fermentative Carbon Metabolism in a Cyanobacterium

Applied and Environmental Microbiology, 2010

Some aquatic microbial oxygenic photoautotrophs (AMOPs) make hydrogen (H 2 ), a carbon-neutral, r... more Some aquatic microbial oxygenic photoautotrophs (AMOPs) make hydrogen (H 2 ), a carbon-neutral, renewable product derived from water, in low yields during autofermentation (anaerobic metabolism) of intracellular carbohydrates previously stored during aerobic photosynthesis. We have constructed a mutant (the ldhA mutant) of the cyanobacterium Synechococcus sp. strain PCC 7002 lacking the enzyme for the NADH-dependent reduction of pyruvate to D-lactate, the major fermentative reductant sink in this AMOP. Both nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography-mass spectrometry (LC-MS) metabolomic methods have shown that autofermentation by the ldhA mutant resulted in no D-lactate production and higher concentrations of excreted acetate, alanine, succinate, and hydrogen (up to 5-fold) compared to that by the wild type.

Research paper thumbnail of Identification and quantification of water-soluble metabolites by cryoprobe-assisted nuclear magnetic resonance spectroscopy applied to microbial fermentation

Magnetic Resonance in Chemistry, 2009

We highlight a range of cryoprobe-assisted NMR methods for studying metabolite production by cyan... more We highlight a range of cryoprobe-assisted NMR methods for studying metabolite production by cyanobacteria, which should be valuable for a wide range of biological applications requiring ultrasensitivity and precise concentration determination over a large dynamic range. Cyroprobe-assisted 1 H and 13 C NMR have been applied to precise determination of metabolic products excreted during autofermentation in two cyanobacterial species: filamentous Arthrospira (Spirulina) maxima CS-328 and unicellular Synechococcus sp. PCC 7002. Several fermentative end products were identified and quantified in concentrations ranging from 50 to 3000 µM in cell-free media (a direct measurement of native-like samples) with less than 5.5% relative error in under 10 min of acquisition per sample with the assistance of an efficient water-suppression protocol. Relaxation times (T1) of these metabolites in aqueous ( 1 H 2 O) solution were measured and found to vary by nearly threefold, necessitating generation of individual calibration curves for each species for highest precision. However, using a 4.5 × longer overall recycle delay between scans, the metabolite concentrations can be predicted within 25% error by calibrating only to a single calibration standard (succinate); other metabolites are then calculated on the basis of their signal integrals and known proton degeneracies. Precise ratios of concentrations of 13 C-labeled versus unlabeled metabolites were determined from integral ratios of 1 H peaks that exhibit 13 C-1 H J-couplings and independently confirmed by direct measurement of areas of corresponding 13 C resonances. 13 C NMR was used to identify and quantify production of osmolytes, trehalose, and glucosylglycerol by A. maxima.