Direct and High-Productive Conversion from a Cyanobacterium Arthrospira Platensis to Ethanol

  In recent years, there is an urgent need to promote the industrial production of biofuels and biochemicals due to environmental issues. However, considering the increasing demand for edible food grains with population expansion, it becomes important to use inedible feedstocks for biorefinery.

  Oxygenic photosynthetic microorganisms such as cyanobacteria or microalgae that accumulate polyglucan such as starch or glycogen, show promise as an alternative feedstock for biorefinery. They have benefits of high-photosynthetic activity and whole-year cultivation without utilizing arable lands. Based on these advantages, the bioethanol production from cyanobacteria or microalgae has been developed in the several previous reports. The microalgal or cyanobacterial polyglucan can be converted to ethanol through saccharification and microbial fermentation. However, the conventional ethanol production process from them composing of pretreatment step and hydrolysis step is high-energy demand and high-environmental load.

  We aim to develop the conversion process with simple, low-energy demand, and low-environmental load from cyanobacteria or microalgae. In cyanobacteria and microalgae, the halophilic cyanobacterium Arthrospira platensis, commonly known as Spirulina, accumulates a large amount of glycogen. In addition, the intracellular glycogen is expected to be extracted relatively simple because of their brittle cell membrane architecture.

  In the present study, to hydrolyze the glycogen in A. platensis without amylases addition, we used a recombinant Saccharomyces cerevisiae strain displaying α-amylase from Streptococcus bovis and glucoamylase from Rhizopus oryzae on the cell surface. As the result, A. platensis could be converted directly to ethanol through a consolidated bioprocess, which is combining glycogen extraction, glycogen saccharification, and ethanol fermentation by using the amylase expressing yeast. However, an ethanol titer (20 g L^-1), an ethanol yield (40% of theoretical yield), and an ethanol productivity (0.3 g L^‒1 h^‒1) were insufficient to accommodate market demand. To improve the ethanol production from A. platensis, the intracellular glycogen has to be more effectively extracted.

  The cell walls in A. platensis such as the peptidoglycan layer and the outer membrane covered with lipopolysaccharide would be barriers to obtain the intracellular glycogen. Lysozyme hydrolyzes cyanobacterial peptidoglycan layer. Additionally, cations may weaken outer membrane because the secretory amount of a recombinant protein from E. coli is increased with the release of lipopolysaccharide by divalent cation such as Ca²⁺ or Mg²⁺. Therefore, to enhance the glycogen extraction from A. platensis, we examined the effect of lysozyme and/or cations on the glycogen extraction. As the result, the ethanol titer and ethanol yield were enhanced by lysozyme addition with an increasing in the total amount of extracted glycogen. The ethanol productivity was accelerated by CaCl₂ addition with a releasing of lipopolysaccharide from outer membrane. Consequently, we successfully developed a direct ethanol conversion process by the amylase expressing yeast from A. platensis with a high titer (48 g L^‒1), a high yield (94% of theoretical yield), and a high productivity (1.0 g L^‒1 h^‒1) by the addition of lysozyme and CaCl₂. The present findings indicate that A. platensis is a remarkable carbohydrate feedstock, which is a promising material for the production of bioethanol and various other commercially valuable chemicals.