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

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.Growth of aquatic microbial oxygenic phototrophs (AMOPs) such as cyanobacteria, algae, and diatoms as renewable feedstocks for energy production has been proposed as an advantageous alternative to growing land-based crops for biofuels (9, 11, 14). These organisms can be grown efficiently on water, sunlight, carbon dioxide, and minimal nutrients on nonarable land or at coastal marine sites. They produce easily digested biopolymers that can be more readily converted to fuels than recalcitrant cellulosic feedstocks. Nevertheless, efficient strategies for converting accumulated biomass from AMOPs into useful fuels are still needed.One strategy for converting cyanobacterial biomass to liquid and gaseous fuels is to allow these cells to rely on their own fermentative pathways—a process termed “autofermentation.” With autofermentation, cells anaerobically catabolize their internally stored carbohydrate molecules (glycogen and soluble sugars), producing CO₂, reductants, and energy as ion gradients and phosphorylation to regenerate ATP. The reducing equivalents in the form of NAD(P)H may be reused by the cell or excreted from the cell as reduced carbon products, typically organic acids and alcohols. The identities of these products are determined by the physiological conditions and by which fermentative enzymes are active. The genomes of cyanobacteria differ in terms of which genes of fermentative enzymes are present and functional, leading to a range of possible fermentative product yields and rates. A major limitation to the technological use of autofermentation for fuel production from biomass is the slow time scale of the conversion in relation to the light/dark cycle of growth.Two fermentation products useful as fuels that are excreted by some cyanobacteria are ethanol and hydrogen. Ethanol production via autofermentation occurs naturally in some cyanobacteria (22). Genetic engineering has been applied successfully to establish autofermentative ethanol production in the cyanobacterium Synechococcus sp. PCC 7942, which does not produce detectable amounts of ethanol in the wild type (10). This strain was created by insertion of the genes for pyruvate decarboxylase and alcohol dehyrogenase from Zymomonas mobilis. Genetic engineering has also been successfully applied to stimulate fermentative hydrogen production in Synechococcus sp. PCC 7002 by increasing the level of the limiting cellular reductant NADH via disruption of the lactate dehydrogenase gene (19).For biotechnological applications, the following two goals have been identified for increasing production of autofermentation products and hydrogen by AMOPs (1): (i) increase photoautotrophic accumulation of stored carbohydrates and (ii) increase the catabolic rate of carbohydrates under dark anaerobic conditions. Here we have continued our work with Arthrospira (Spirulina) maxima with efforts to achieve these two goals.Different approaches for increasing carbohydrate content for cyanobacteria exist. One is nutrient deprivation. Many cyanobacteria, including Arthrospira species, do not have nitrogenase (nif) genes and therefore require a nitrogen source (such as nitrate, ammonia, or urea) for protein synthesis. Deprivation of a nitrogen source is a well-documented strategy for increasing the glycogen content (stored as insoluble carbohydrate granules) in many nondiazotrophic cyanobacteria (3, 13, 23, 26). Sulfur deprivation has also been shown to increase the glycogen content in at least two cyanobacteria when incubated in the presence of methane (2). Recently, it was demonstrated that sulfur and nitrogen limitation, rather than complete deprivation, provides optimal autofermentative hydrogen yields in the cyanobacterium Synechocystis sp. PCC 6803 (5).A different approach, which increases soluble sugars in cyanobacteria, involves adaptation of cells to media with high concentrations of sodium salts. Many cyanobacteria accumulate organic molecules such as glucosyl-glycerol and/or trehalose to osmotically balance their cytosols with the extracellular medium (6, 17, 20). In addition to glycogen, these molecules can serve as substrates for fermentation in cyanobacteria (22). Both glucosyl-glycerol and trehalose are present in Arthrospira platensis (29) and A. maxima CS-328 (8). It was shown that a 4-fold increase in carbohydrate content can be achieved by growing A. platensis in media supplemented with additional 1 M NaCl relative to no additional NaCl (28).Acceleration of carbohydrate autofermentation in cyanobacteria by application of selective physiological stresses has been previously proposed (1). Here we report a new strategy that combines the established method for increasing carbohydrate content in Arthrospira by adapting filaments to highly saline growth media (28) with hypoionic stress to accelerate autofermentation. The entire process can be described as “sodium stress cycling,” which relies on hyperionic conditions (high salt) during growth to accumulate sugars, followed by hypoionic stress (low salt) to force catabolism during autofermentation. By resuspending filaments grown in media containing excess NaCl into buffer containing only sufficient solute to prevent lysis at the start of autofermentation, we achieve a large increase in total fermentative product yields relative to cells that were not adapted to high salt. However, this strategy did not lead to higher hydrogen yields, demonstrating that other fermentative routes (primarily ethanol production) are used for NADH recycling in A. maxima under hypoionic conditions.