Abstract
Although significant advances in H2 photoproduction have recently been realized in fresh water algae (e.g. Chlamydomonas reinhardtii), relatively few studies have focused on H2 production and hydrogenase adaptations in marine or halophilic algae. Salt water organisms likely offer several advantages for biotechnological H2 production due to the global abundance of salt water, decreased H2 and O2 solubility in saline and hypersaline systems, and the ability of extracellular NaCl levels to influence metabolism. We screened unialgal isolates obtained from hypersaline ecosystems in the southwest United States and identified two distinct halophilic strains of the genus Tetraselmis (GSL1 and QNM1) that exhibit both robust fermentative and photo H2-production activities. The influence of salinity (3.5%, 5.5% and 7.0% w/v NaCl) on H2 production was examined during anoxic acclimation, with the greatest in vivo H2-production rates observed at 7.0% NaCl. These Tetraselmis strains maintain robust hydrogenase activity even after 24 h of anoxic acclimation and show increased hydrogenase activity relative to C. reinhardtii after extended anoxia. Transcriptional analysis of Tetraselmis GSL1 enabled sequencing of the cDNA encoding the FeFe-hydrogenase structural enzyme (HYDA) and its maturation proteins (HYDE, HYDEF and HYDG). In contrast to freshwater Chlorophyceae, the halophilic Tetraselmis GSL1 strain likely encodes a single HYDA and two copies of HYDE, one of which is fused to HYDF. Phylogenetic analyses of HYDA and concatenated HYDA, HYDE, HYDF and HYDG in Tetraselmis GSL1 fill existing knowledge gaps in the evolution of algal hydrogenases and indicate that the algal hydrogenases sequenced to date are derived from a common ancestor. This is consistent with recent hypotheses that suggest fermentative metabolism in the majority of eukaryotes is derived from a common base set of enzymes that emerged early in eukaryotic evolution with subsequent losses in some organisms.
Highlights
The phylogenetically unrelated NiFe- and FeFe-hydrogenases have convergently evolved to catalyze the reversible reduction of protons to H2 (2H++2e2, = .H2) [1]
To assess whether algae with hydrogenase activity are present in hypersaline environments, water samples were collected from a variety of Great Salt Lake (GSL) sites with salinities ranging from 3.5–25%, and unialgal isolates from these water samples were obtained using flow cytometry
Hydrogenase activity screens of over 40 unialgal isolates recovered from GSL using the reduced methyl viologen (MV) assay revealed that the most robust hydrogenase activity was detected from a tetraflagellate alga that morphology and 18S rRNA gene analysis indicated to be a novel strain of Tetraselmis (Tetraselmis strain GSL1), isolated from a roadside pool bordering GSL with a salinity of 6.0% w/v
Summary
Several eukaryotic algae generate fermentative H2 during dark, anoxic acclimation as part of a suite of fermentative pathways that catabolize carbohydrates to alcohols, organic acids and H2, which are secreted. These metabolites likely provide a rich source of carbon building blocks and reducing equivalents to organisms inhabiting ecological niches adjacent to the algae, which are responsible for the majority of primary productivity during the day. Only FeFe-hydrogenases have been unambiguously identified in algae, with organisms such as Chlamydomonas reinhardtii encoding truncated enzymes with only the catalytic H-cluster; a 4Fe4S cluster linked via a bridging cysteine to a two Fe center coordinated by CN2/CO ligands and a bridging dithiolate. Chlorella variabilis NC64A encodes two hydrogenase enzymes with both the H-cluster catalytic domain and an F-cluster domain that coordinates additional FeS clusters that putatively function in electron transfer [18], [19], [24,25,26]
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