Damian Carrieri

Aquatic phototrophs: efficient alternatives to land-based crops for biofuels

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.

Optimization of Metabolic Capacity and Flux through Environmental Cues To Maximize Hydrogen Production by the Cyanobacterium “Arthrospira (Spirulina) maxima”

ABSTRACT Environmental and nutritional conditions that optimize the yield of hydrogen (H2) from water using a two-step photosynthesis/fermentation (P/F) process are reported for the hypercarbonate-requiring cyanobacterium “Arthrospira maxima.” Our observations lead to four main conclusions broadly applicable to fermentative H2 production by bacteria: (i) anaerobic H2 production in the dark from whole cells catalyzed by a bidirectional [NiFe] hydrogenase is demonstrated to occur in two temporal phases involving two distinct metabolic processes that are linked to prior light-dependent production of NADPH (photosynthetic) and dark/anaerobic production of NADH (fermentative), respectively; (ii) H2 evolution from these reductants represents a major pathway for energy production (ATP) during fermentation by regenerating NAD+ essential for glycolysis of glycogen and catabolism of other substrates; (iii) nitrate removal during fermentative H2 evolution is shown to produce an immediate and large stimulation of H2, as nitrate is a competing substrate for consumption of NAD(P)H, which is distinct from its slower effect of stimulating glycogen accumulation; (iv) environmental and nutritional conditions that increase anaerobic ATP production, prior glycogen accumulation (in the light), and the intracellular reduction potential (NADH/NAD+ ratio) are shown to be the key variables for elevating H2 evolution. Optimization of these conditions and culture age increases the H2 yield from a single P/F cycle using concentrated cells to 36 ml of H2/g (dry weight) and a maximum 18% H2 in the headspace. H2 yield was found to be limited by the hydrogenase-mediated H2 uptake reaction.

Boosting Autofermentation Rates and Product Yields with Sodium Stress Cycling: Application to Production of Renewable Fuels by Cyanobacteria

ABSTRACT Sodium concentration cycling was examined as a new strategy for redistributing carbon storage products and increasing autofermentative product yields following photosynthetic carbon fixation in the cyanobacterium Arthrospira (Spirulina) maxima. The salt-tolerant hypercarbonate strain CS-328 was grown in a medium containing 0.24 to 1.24 M sodium, resulting in increased biosynthesis of soluble carbohydrates to up to 50% of the dry weight at 1.24 M sodium. Hypoionic stress during dark anaerobic metabolism (autofermentation) was induced by resuspending filaments in low-sodium (bi)carbonate buffer (0.21 M), which resulted in accelerated autofermentation rates. For cells grown in 1.24 M NaCl, the fermentative yields of acetate, ethanol, and formate increase substantially to 1.56, 0.75, and 1.54 mmol/(g [dry weight] of cells·day), respectively (36-, 121-, and 6-fold increases in rates relative to cells grown in 0.24 M NaCl). Catabolism of endogenous carbohydrate increased by approximately 2-fold upon hypoionic stress. For cultures grown at all salt concentrations, hydrogen was produced, but its yield did not correlate with increased catabolism of soluble carbohydrates. Instead, ethanol excretion becomes a preferred route for fermentative NADH reoxidation, together with intracellular accumulation of reduced products of acetyl coenzyme A (acetyl-CoA) formation when cells are hypoionically stressed. In the absence of hypoionic stress, hydrogen production is a major beneficial pathway for NAD+ regeneration without wasting carbon intermediates such as ethanol derived from acetyl-CoA. This switch presumably improves the overall cellular economy by retaining carbon within the cell until aerobic conditions return and the acetyl unit can be used for biosynthesis or oxidized via respiration for a much greater energy return.

The role of the bidirectional hydrogenase in cyanobacteria

Cyanobacteria have tremendous potential to produce clean, renewable fuel in the form of hydrogen gas derived from solar energy and water. Of the two cyanobacterial enzymes capable of evolving hydrogen gas (nitrogenase and the bidirectional hydrogenase), the hox-encoded bidirectional Ni-Fe hydrogenase has a high theoretical potential. The physiological role of this hydrogenase is a highly debated topic and is poorly understood relative to that of the nitrogenase. Here the structure, assembly, and expression of this enzyme, as well as its probable roles in metabolism, are discussed and analyzed to gain perspective on its physiological role. It is concluded that the bidirectional hydrogenase in cyanobacteria primarily functions as a redox regulator for maintaining a proper oxidation/reduction state in the cell. Recommendations for future research to test this hypothesis are discussed.

Photo-catalytic conversion of carbon dioxide to organic acids by a recombinant cyanobacterium incapable of glycogen storage

Deletion of the gene encoding glucose-1-phosphate adenylyltransferase (ΔglgC) in the non-nitrogen-fixing cyanobacterium, Synechocystis sp. PCC 6803, disables glycogen synthesis, arrests cellular biomass accumulation under nitrogen deficiency, and redirects photosynthetically fixed carbon to organic acids (α-ketoglutarate and pyruvate) that appear in the extracellular medium.

Genetic Analysis of the Hox Hydrogenase in the Cyanobacterium Synechocystis sp. PCC 6803 Reveals Subunit Roles in Association, Assembly, Maturation, and Function

Background: Hox hydrogenase (HoxEFUYH) is present in some bacteria and cyanobacteria. Results: In Synechocystis, HoxYH and HoxEFU form subcomplexes that can be disrupted in Hox subunit mutants. Conclusion: Hox assembly is modular and most reliant on the presence of HoxF and -U, consistent with effects on Hox protein abundance and activity. Significance: Subcomplexes of Hox hydrogenase may represent steps in complex assembly and maturation. Hydrogenases are metalloenzymes that catalyze 2H+ + 2e− ↔ H2. A multisubunit, bidirectional [NiFe]-hydrogenase has been identified and characterized in a number of bacteria, including cyanobacteria, where it is hypothesized to function as an electron valve, balancing reductant in the cell. In cyanobacteria, this Hox hydrogenase consists of five proteins in two functional moieties: a hydrogenase moiety (HoxYH) with homology to heterodimeric [NiFe]-hydrogenases and a diaphorase moiety (HoxEFU) with homology to NuoEFG of respiratory Complex I, linking NAD(P)H ↔ NAD(P)+ as a source/sink for electrons. Here, we present an extensive study of Hox hydrogenase in the cyanobacterium Synechocystis sp. PCC 6803. We identify the presence of HoxEFUYH, HoxFUYH, HoxEFU, HoxFU, and HoxYH subcomplexes as well as association of the immature, unprocessed large subunit (HoxH) with other Hox subunits and unidentified factors, providing a basis for understanding Hox maturation and assembly. The analysis of mutants containing individual and combined hox gene deletions in a common parental strain reveals apparent alterations in subunit abundance and highlights an essential role for HoxF and HoxU in complex/subcomplex association. In addition, analysis of individual and combined hox mutant phenotypes in a single strain background provides a clear view of the function of each subunit in hydrogenase activity and presents evidence that its physiological function is more complicated than previously reported, with no outward defects apparent in growth or photosynthesis under various growth conditions.

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

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 13C1H 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

Engineered xylose utilization enhances bio-products productivity in the cyanobacterium Synechocystis sp. PCC 6803.

Hydrolysis of plant biomass generates a mixture of simple sugars that is particularly rich in glucose and xylose. Fermentation of the released sugars emits CO2 as byproduct due to metabolic inefficiencies. Therefore, the ability of a microbe to simultaneously convert biomass sugars and photosynthetically fix CO2 into target products is very desirable. In this work, the cyanobacterium, Synechocystis 6803, was engineered to grow on xylose in addition to glucose. Both the xylA (xylose isomerase) and xylB (xylulokinase) genes from Escherichia coli were required to confer xylose utilization, but a xylose-specific transporter was not required. Introduction of xylAB into an ethylene-producing strain increased the rate of ethylene production in the presence of xylose. Additionally, introduction of xylAB into a glycogen-synthesis mutant enhanced production of keto acids. Isotopic tracer studies found that nearly half of the carbon in the excreted keto acids was derived from the engineered xylose metabolism, while the remainder was derived from CO2 fixation.

Contribution of a sodium ion gradient to energy conservation during fermentation in the cyanobacterium Arthrospira (Spirulina) maxima CS-328.

ABSTRACT Sodium gradients in cyanobacteria play an important role in energy storage under photoautotrophic conditions but have not been well studied during autofermentative metabolism under the dark, anoxic conditions widely used to produce precursors to fuels. Here we demonstrate significant stress-induced acceleration of autofermentation of photosynthetically generated carbohydrates (glycogen and sugars) to form excreted organic acids, alcohols, and hydrogen gas by the halophilic, alkalophilic cyanobacterium Arthrospira (Spirulina) maxima CS-328. When suspended in potassium versus sodium phosphate buffers at the start of autofermentation to remove the sodium ion gradient, photoautotrophically grown cells catabolized more intracellular carbohydrates while producing 67% higher yields of hydrogen, acetate, and ethanol (and significant amounts of lactate) as fermentative products. A comparable acceleration of fermentative carbohydrate catabolism occurred upon dissipating the sodium gradient via addition of the sodium-channel blocker quinidine or the sodium-ionophore monensin but not upon dissipating the proton gradient with the proton-ionophore dinitrophenol (DNP). The data demonstrate that intracellular energy is stored via a sodium gradient during autofermentative metabolism and that, when this gradient is blocked, the blockage is compensated by increased energy conversion via carbohydrate catabolism.

Enhancing photo‐catalytic production of organic acids in the cyanobacterium Synechocystis sp. PCC 6803 ΔglgC, a strain incapable of glycogen storage

A key objective in microbial biofuels strain development is to maximize carbon flux to target products while minimizing cell biomass accumulation, such that ideally the algae and bacteria would operate in a photo‐catalytic state. A brief period of such a physiological state has recently been demonstrated in the cyanobacterium Synechocystis sp. PCC 6803 ΔglgC strain incapable of glycogen storage. When deprived of nitrogen, the ΔglgC excretes the organic acids alpha‐ketoglutarate and pyruvate for a number of days without increasing cell biomass. This study examines the relationship between the growth state and the photo‐catalytic state, and characterizes the metabolic adaptability of the photo‐catalytic state to increasing light intensity. It is found that the culture can transition naturally from the growth state into the photo‐catalytic state when provided with limited nitrogen supply during the growth phase. Photosynthetic capacity and pigments are lost over time in the photo‐catalytic state. Reversal to growth state is observed with re‐addition of nitrogen nutrient, accompanied by restoration of photosynthetic capacity and pigment levels in the cells. While the overall productivity increased under high light conditions, the ratio of alpha‐ketoglutarate/pyruvate is altered, suggesting that carbon partition between the two products is adaptable to environmental conditions.