Defluviitalea phaphyphila sp. nov., a Novel Thermophilic Bacterium That Degrades Brown Algae

Abstract

Brown algae are one of the largest groups of oceanic primary producers for CO 2 removal and carbon sinks for coastal regions. However, the mechanism for brown alga assimilation remains largely unknown in thermophilic microorganisms. In this work, a thermophilic alginolytic community was enriched from coastal sediment, from which an obligate anaerobic and thermophilic bacterial strain, designated Alg1, was isolated. Alg1 shared a 16S rRNA gene identity of 94.6% with Defluviitalea saccharophila LIND6LT2 T . Phenotypic, chemotaxonomic, and phylogenetic studies suggested strain Alg1 represented a novel species of the genus Defluviitalea, for which the name Defluviitalea phaphyphila sp. nov. is proposed. Alg1 exhibited an intriguing ability to convert carbohydrates of brown algae, including alginate, laminarin, and mannitol, to ethanol and acetic acid. Three gene clusters participating in this process were predicted to be in the genome, and candidate enzymes were successfully expressed, purified, and characterized. Six alginate lyases were demonstrated to synergistically deconstruct alginate into unsaturated monosaccharide, followed by one uronic acid reductase and two 2-keto-3-deoxy-D-gluconate (KDG) kinases to produce pyruvate. A nonclassical mannitol 1-phosphate dehydrogenase, catalyzing D-mannitol 1-phosphate to fructose 1-phosphate in the presence of NAD ؉ , and one laminarase also were disclosed. This work revealed that a thermophilic brown alga-decomposing system containing numerous novel thermophilic alginate lyases and a unique mannitol 1-phosphate dehydrogenase was adopted by the natural ethanologenic strain Alg1 during the process of evolution in hostile habitats. Brown algae (Phaeophyceae) are a large group of marine vegetation that have abundant amounts of the yellow-brown pigment fucoxanthin (1). Their huge biomass makes them one of the largest oceanic primary producers for CO 2 removal and carbon storage for coastal regions (2). Moreover, as an emerging feedstock for liquid biofuel production, recently the bioconversion of brown algae has been reported frequently (1, 3-6). They have a complex sugar composition, mainly including alginate, mannitol, and laminarin (6). Alginate is a unique structural polysaccharide in brown algae, and it is abundant in the cell wall for mechanical protection (1). It is a linear polysaccharide consisting of two uronic acids, ␣-L-guluronate (G) and ␤-D-mannuronate (M) (7). The content of alginate varies by species from 20% to 40% dry cell weight (8, 9). Mannitol is a sugar alcohol form of mannose. Laminarin, a storage polysaccharide in many brown algae, is a linear polysaccharide of ␤-1,3-linked glucose with small amounts of ␤-1,6-linkages (10). Mannitol and laminarin are mostly accumulated in the summer, and their content could reach as high as 25% and 30% in the species Laminaria hyperborean (11).The microbial degradation of brown algae involves the decomposition of the structural polysaccharides (mainly alginate and laminarin) and then the catabolism of the resulting mon...

A thermostable mannitol‐1‐phosphate dehydrogenase is required in mannitol metabolism of the thermophilic acetogenic bacterium Thermoanaerobacter kivui

“…is also evident, with 58%–62% sequence identity. As described previously, thermophiles have been described to utilize mannitol (Ji et al ., ; Chades et al ., ), and other thermostable MtlDs are awaiting their biological characterization. For the acetogens, it remains to be elucidated whether more acetogens utilize mannitol, and whether MtlD is important for mannitol oxidation in these species.…”

“…In other thermophiles, different enzymes may be involved in mannitol metabolism. For example, in the thermophilic Clostridia-related species Defluviitalea phaphyphila (Ji et al, 2016b), fructose-1-phosphate, not fructose-6-phosphate, is the product of mannitol-1-phosphate oxidation. The specific activity of the responsible MtlD is orders of magnitudes lower than that of T. kivui MtlD (4.3 μmol min −1 mg −1 ).…”

“…The sugar alcohol mannitol is of environmental and of biotechnological interest, because it is produced and stored as a reserve carbohydrate in brown algae during the summer months with concentrations of greater than 30% on a weight basis (Adams et al, 2011). Mannitol serves as substrate for growth and energy conservation for many bacteria and yeasts, among these are obligate anaerobic thermophiles within the class Clostridia (Ji et al, 2016b;Chades et al, 2018), a phylum that also harbours many acetogenic species (Drake et al, 2008). Recently, direct conversion of brown algae biomass high in mannitol and laminarin to ethanol was observed in the thermophilic bacterium Defluviitalea phaphyphila (Ji et al, 2016a), emphasizing the potential of brown algae biomass as a renewable energy source.…”

Microbial Community Drivers in Anaerobic Granulation at High Salinity

“…The species type of the family, Defluviitalea saccharophila, was isolated from an UASB reactor (Jabari et al, 2012). Defluviitalea phaphyphila, isolated from coastal sediment, is the only strain able to tolerate up to 5% NaCl (w/v), but not able to grow on proteins as casaminoacids (Ji et al, 2016). Thus, a new mesophilic species was represented in this study, with high salinity tolerance and most likely involved in protein fermentation and/or EPS production.…”

Substitution of one calcium‐binding amino acid strengthens substrate binding in a thermophilic alginate lyase

“…E. coli was cultured in LB medium (10 gÁL À1 NaCl, 10 gÁL À1 tryptone, and 5 gÁL À1 yeast extract) supplemented with 100 lgÁmL À1 ampicillin when required. D. phaphyphila (=CGMCC 1.5199 T = JCM 30481 T ) was cultured at 60°C according to previous report (Ji et al 2015).…”