Hydrogen Evolution as a Consumption Mode of Reducing Equivalents in Green Algal Fermentation

Abstract

Dark anaerobic fermentation in the green algae Chiamydomonas MGA 161, Chlamydomonas reinhardtii, Chlorella pyrenoidosa, and Chlorococcum minutum was studied. Our isolate, Chiamydomonas MGA 161, was unusual in having high H2 but almost no formate. The fermentation pattern in Chlamydomonas MGA 161 was altered by changes in the NaCI or NHI-CI concentration. Glycerol formation increased at low (0.1%) and high (7%) NaCI concentrations; starch degradation, and formation of ethanol, H2, and CO2 increased with the addition of NH4CI to above 5 millimolar in N-deficient cells. C. reinhardtii and C. pyrenoidosa exhibited a very similar anaerobic metabolism, forming formate, acetate and ethanol in a ratio of about 2:2:1. C. minutum was also unusual in forming acetate, glycerol, and CO2 as its main products, with H2, formate, and ethanol being formed in negligible amounts. In the presence of CO, ethanol formation increased twofold in Chiamydomonas MGA 161 and C. reinhardtii, but the fermentation pattern in C. minutum did not change. An experiment with hypophosphite addition showed that dark H2 evolution of the Escherichia coli type could be ruled out in Chiamydomonas MGA 161 and C. reinhardtii. Among the green algae investigated, three fermentation types were identified by the distribution pattern of the end products, which reflected the consumption mode of reducing equivalents in the cells.Green algae produce hydrogen under dark anaerobic conditions, as first observed by Gaffron and Rubin (8). The whole process of dark H2 evolution is mediated through the fermentative degradation of accumulated starch. Use of this property in a stable biophotolysis system makes possible temporal separation of H2 production from photosynthetic 02 production. Lightdependent H2 production without an O2 defense system soon comes to a stop because of inhibition by the O2 produced (10, 28). Studies with a freshwater alga, Chlamydomonas reinhardtii, showed an intermittent but stable 02/H2 production system in an alternating light/dark cycle (24,25). A halotolerant green alga, Chlamydomonas MGA 161, has been isolated, and both growth and dark H2 evolution in this alga are active (26 ucts of green algae (9,17,19). Klein and Betz (16) reported on the fermentation metabolism with no formate formation in Chlamydomonas moewusii. Gfeller and Gibbs (9) and Kreuzberg (19) reported that formate, acetate, and ethanol are produced in a ratio of 2:1:1 in the dark incubation of C. reinhardtii. The fermentation process is regulated by ATP and reducing equivalents generated during starch degradation. The ATP concentration within Chlorella cells increases slightly with active hydrogenase under dark anaerobic conditions, accompanied by an increase in NADH and a decrease in NADPH (22). Reducing equivalents are consumed by the formation of fermentation products for a constant metabolic flux; the reducing power is used for the formation of H2 (from H+), formate (from C02), glycerol (from acetonephosphate) or ethanol (from acetyl-CoA) (17).In this paper, we rep...

Microalgal triacylglycerols as feedstocks for biofuel production: perspectives and advances

“…Chlamydomonas reinhardtii can also be used as a model organism for fermentation, given the number of pathways identified under anaerobic conditions biochemically (Gfeller and Gibbs, 1984;Ohta et al, 1987) or by microarray analysis (Mus et al, 2007). The results, summarized in Figure 4, suggest that both the pyruvate formate lyase (PFL) and the pyruvate ferredoxin oxidoreductase (PFR) pathways are functional in C. reinhardtii under anaerobiosis, as well as the pyruvate decarboxylase (PDC) pathway.…”

A Mutant in theADH1Gene ofChlamydomonas reinhardtiiElicits Metabolic Restructuring during Anaerobiosis

“…The anoxic environment elicits the synthesis/activation of enzymes that ferment sugars, producing organic acids, ethanol, CO 2 , and H 2 (Gfeller and Gibbs, 1984;Kreuzberg, 1984;Ohta et al, 1987). We and others are developing Chlamydomonas as a model system to elucidate pathways and regulatory circuits associated with fermentation metabolism in photosynthetic, eukaryotic microbes.…”

“…It relies on glycolytic breakdown of carbohydrate reserves and activation of fermentation pathways for generating the energy required for survival during periods of oxygen depletion (Gfeller and Gibbs, 1984;Kreuzberg, 1984;Ohta et al, 1987). A number of these fermentation pathways are typical of those present in various prokaryotes and some eukaryotes (Mus et al, 2007).…”

“…Some enzymes that function in these pathways include pyruvate:ferredoxin oxidoreductase (PFR), pyruvate decarboxylase (PDC), lactate dehydrogenase (LDH), pyruvate formate lyase (PFL), alcohol dehydrogenase (ADH), phosphate acetyltransferase (PAT), acetate kinase (ACK), and the two [FeFe] hydrogenases (HYDA1 and HYDA2) and their maturation proteins, HYDG and HYDEF (Posewitz et al, 2004;Atteia et al, 2006;Ghirardi et al, 2007;Mus et al, 2007;Hemschemeier et al, 2008;Grossman et al, 2011). The anaerobic activities of these and other enzymes result in the secretion of organic acids (formate, lactate, malate, acetate, and succinate) and alcohols (ethanol and glycerol) as well as the evolution of H 2 and CO 2 (Gfeller and Gibbs, 1984;Kreuzberg, 1984;Ohta et al, 1987;Tsygankov et al, 2002;Kosourov et al, 2003;Mus et al, 2007;Dubini et al, 2009).…”

“…Under the fermentation conditions used, ethanol accounts for about one-fourth of the metabolites synthesized and excreted by Chlamydomonas cells during dark anoxia (Gfeller and Gibbs, 1984;Kreuzberg, 1984;Ohta et al, 1987). Cytoplasmic PDC and ADH activities typically ferment pyruvate to ethanol in terrestrial plants (Dolferus et al, 1985;Mü cke et al, 1995;Kü rsteiner et al, 2003; for review, see Magneschi and Perata, 2009).…”