Production and characterization of bio-based surfactants from Bacillus subtilis and Pseudomonas aeruginosa grown on crude sugar hydrolysates from low-value fibrous biomasses.

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2022-05
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Sharma, Rajat
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Lamsal, Buddhi P
Halverson, Larry J
Wang, Tong
Wright, Mark M
Wen, Zhiyou
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Food Science and Human Nutrition
Abstract
Bio-based surfactants have shown immense potential as environment-friendly alternatives to petroleum-based surfactants for many food and non-food applications. Projected at a global market value of $5.52 billion in 2022, bio-based surfactants have been studied extensively, and many research studies have focused on their production strategies, downstream processing, and functional characterization. The overarching goal of this study was to investigate the use of important low-cost fibrous biomasses as carbohydrate sources for the production of biosurfactants from two bacterial strains, a) Bacillus subtilis and b) Pseudomonas aeruginosa, downstream processing/ separation, and characterization of important properties. These goals were accomplished by 1) optimizing alkali pretreatment (liquid ammonia) and ultrasonication parameters for six fibrous biomasses, a) switchgrass, b) alfalfa, c) soy hulls, d) soy fiber, e) dry distillers’ grain with solubles (DDGS), and f) bagasse; 2) assessing growth and bio-based surfactant production performance of Bacillus and Pseudomonas strains on crude biomass hydrolysate based optimized media and bio-enhancers; and 3) characterizing isoforms of purified surfactin and testing their marine toxicity and surface activity. In the studies associated with optimizing pretreatment parameters for fibrous biomasses and testing the growth performance on Bacillus strains, it was found that switchgrass and alfalfa hydrolysate based media provided comparable or better growth than pure glucose-based media; however, soy hull hydrolysate based media yielded surfactant titer performance at par with pure glucose media (2.9 g/L and 0.28 g/L for surfactin and FA-Glu) in 50-mL shake flask studies which were verified at 5-L fermentation. A simple techno-economic analysis for the cost of production of surfactin on soy-hull-based media through simulation of a 250-L fermentation facility processing utilizing 15 kg soy hulls in a batch with a plant-biomass processing capacity of 2000 tonne dry biomass /day, indicated the cost to be $6.63/kg for 97% pure product. Soy hull based hydrolysates were utilized with three other pure sugar controls to investigate the growth of Pseudomonas aeruginosa and rhamnolipid titer production along with bio-enhancers (norepinephrine and dopamine). It was shown that soy hulls supported the production of rhamnolipids (1.98 g/L) although less efficiently compared to glucose and glycerol controls (2.78 g/L and 2.72 g/L). No significant effect of bio-enhancers was found on log-growth rate during exponential phase or final rhamnolipid titer performance as witnessed in other studies conducted on Pseudomonas aeruginosa. An analytical scale method of surfactin isoforms separation with molecular weights of 992.6, 1006.6, 1020.6, 1034.6, 1048.6, 1062.6 m/z amu was adopted and evaluated with a preparatory HPLC. These isoforms were tested for toxicity on Fundulus grandis, and surface tension profiles were compared. These isoforms were also studied for the effect of crude biomass sugar hydrolysates in growth media on distribution ratios of isoforms. The surface tension values of all these isoforms, in the absence of electrolytes and at 12 ppt salinity, were found to be statistically similar at 37 (pH 6.5) and 31 (pH 9.5) mN/m and the dispersant-to-oil ratio of 1:20. The LC50 for surfactin isoforms against Fundulus grandis ranged between 10 and 20 mg/L, compared to the LC50 values of ~100 mg/L for a variant, an unseparated FA-Glu (Fatty acid Glutamate) at a molecular weight of 344.2 amu- 414.28 amu, thus was deemed least toxic. In summary, our study resulted in crucial technical information on the use of crude sugar hydrolysates from low-value fibrous biomass as bacterial growth media for commercially and functionally important bio-based surfactants, i.e., surfactin and its variant FA-Glu, and rhamnolipid. This study provides a framework for an environmentally sustainable method of production of two important bio-based surfactants for a techno-economic assessment to improve the economic viability of surfactin production.
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