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 (H 2 ) 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 H 2 production by bacteria: (i) anaerobic H 2 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) H 2 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 H 2 evolution is shown to produce an immediate and large stimulation of H 2 , 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 H 2 evolution. Optimization of these conditions and culture age increases the H 2 yield from a single P/F cycle using concentrated cells to 36 ml of H 2 /g (dry weight) and a maximum 18% H 2 in the headspace. H 2 yield was found to be limited by the hydrogenase-mediated H 2 uptake reaction.

Abundance and Diversity of the Phototrophic Microbial Mat Communities of Sulphur Mountain Banff Springs and Their Significance to the Endangered Snail, <i>Physella johnsoni</i>

“…The dominant cyanobacteria resembling Spirulina (Figure 3(F)) proved to be atypical regarding culturing. Contrary to approaches and media published for Spirulina and Arthrospira cultivation [50]- [52], the species at Banff springs preferred neutral over basic pH and perished immediately in widely used Zarrouk-based medium.…”

Synechococcus sp. Strain PCC 7002 nifJ Mutant Lacking Pyruvate:Ferredoxin Oxidoreductase

“…This has previously been shown to occur in two temporally resolved phases in fermenting cells derived from exponentially growing cultures (1). Because a respiratory incubation period prior to fermentation eliminates the first phase of H 2 production in A. maxima, this transient phase had been attributed to accumulated photosynthetic reductant (Fd, NADPH) that is exhausted during respiration.…”

“…Activity was determined immediately following the onset of fermentative conditions. Dissolved H 2 concentrations were measured using a membrane-covered rate (Clark-type) electrode as described previously (1), except that cultures were allowed to ferment directly on the membrane at 35°C. Total sugars were assayed using anthrone reagent (13) as described previously (25).…”

Analysis of carbohydrate storage granules in the diazotrophic cyanobacterium Cyanothece sp. PCC 7822

“…The 99 growth medium used for cultivation of many cyanobacteria, BG-11 (Allen 1968), is a 100 relatively rich media for phototrophic organisms and provides nitrate and phosphate in 101 excess (Ananyev et al 2008; Burrows et al 2008;Chen et al 2008). The molar nitrate 102 and phosphate ratio is much higher than the canonical Redfield ratio (16:1) (Ernst et al 103 2005), as well as the proposed range of nitrate:phosphate (N:P) appropriate for nitrogen 104 fixing species suggested by (Klausmeier et al 2004).…”