Biotechnological processes for efficient resource recovery from residual materials rely on complex conversions carried out by reactor microbiomes. Chain elongation microbiomes produce valuable medium-chain carboxylates (MCC) that can be used as biobased starting materials in the chemical, agriculture and food industry. In this study, sunflower oil is used as an application-compatible solvent to accumulate microbially produced MCC during extractive lactate-based chain elongation. The MCC-enriched solvent is harvested as a potential novel product for direct application without further MCC purification, e.g., direct use for animal nutrition. Sunflower oil biocompatibility, in situ extraction performance and effects on chain elongation were evaluated in batch and continuous experiments. Microbial community composition and dynamics of continuous experiments were analyzed based on 16S rRNA gene sequencing data. Potential applications of MCC-enriched solvents along with future research directions are discussed.
Vegetable oils are envisioned here as promising matrixes to accumulate MCC during extractive chain elongation for the direct application of MCC-enriched oils after being harvested or skimmed off the reactor. Extractive fermentation with application-compatible solvents can lead to novel products from microbial chain elongation while impacting the process positively by, for instance, increasing fermentation efficiency and reducing downstream processing (DSP) complexity. Typically, further steps are needed for solvent regeneration and MCC purification after MCC are concentrated [19]. Solvent regeneration is usually done through back-extraction with strong inorganic bases which requires additional chemicals and generates waste inorganic salts [12]. Purification may be done by energy-demanding processes, such as distillation [20] or membrane electrolysis [19], adding to DSP complexity. At industrial scale, even a six-steps down stream processing (DSP) based on physical separation and evaporation techniques was proposed by ChainCraft B.V. to recover MCC salts obtained from food waste and ethanol for animal nutrition applications [21]. Alternatively, application-compatible solvents to avoid product-solvent separation has been deemed attractive for advanced biofuels fermentation processes [22] with benefits including reduced production costs and environmental footprint [23]. One potential application of MCC-enriched vegetable oils is their use as novel food or feed additives with diverse functionalities. MCC display differential effects on human health compared to unsaturated LCC [24]. Vegetable oils and the LCC contained in them are shown to have positive effects in livestock growth [25, 26], while MCC can be used in low doses to inactivate pathogens in feed and improve swine health and performance [27]. Both LCC and MCC can be used as natural alternatives to antibiotics [26, 27] and for methane mitigation in cattle [28,29,30].
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Thus, this work aims to assess the feasibility of producing MCC-enriched vegetable oil via lactate-based chain elongation. Extraction capability and biocompatibility of sunflower oil, a widely available vegetable oil, is compared against oleyl alcohol in batch extractive fermentation using lactate and food waste as substrates. Then, continuous bioreactor experiments were performed to evaluate extractive chain elongation of l-lactate and acetate and MCC accumulation in sunflower oil. Changes in microbiome composition and taxa differential abundance were studied using 16S rRNA gene sequencing data. MCC extraction with sunflower oil was compared using a synthetic effluent in abiotic continuous reactors.
Using microbially produced carboxylates from low-value organics is getting increasing attention as a way to valorize the growing amounts of residues [53]. Lactate-based chain elongation is promising for upcycling complex organic residues to MCC without adding external electron donors [4, 5]. Here we show that chain elongation from lactate can yield high MCC selectivities. When carboxylates are used as feed additives, livestock-related concerns such as increasing antibiotic resistance [26, 27] and greenhouse gases emissions [30] can be addressed as well. MCC (in their salt form) produced via chain elongation from organic residual materials are now commercially available for animal nutrition [54]. In addition, vegetable oils are also reported to positively influence livestock [25, 26] and they can be used as application-compatible solvents for in situ MCC extraction in chain elongation processes. The use of solvents that are compatible with a specific application may reduce additional equipment, purification steps and salt waste generation. Some of these benefits have been proposed to be achieved in extractive fermentation with engineered strains, where product-enriched solvents are directly used in chemical hydrogenation for aviation fuel applications [22] and reduce process production costs and environmental impact [23]. The potential benefits of application-compatible solvents for the process proposed here should be evaluated considering sustainability aspects related to vegetable oil production [55] and chain elongation processes [56]. Food waste-derived oil could be used as an alternative endogenous solvent that seems to extract MCC [4]. Hazardous compounds possibly present in waste-derived oils [57] may be removed for feed applications. A potential interesting option is to use microbial oils from algae and/or yeast cultures which feature high unsaturated LCC contents and can be obtained using agro-industrial waste streams [58]. Especial attention to extraction of unwanted compounds (e.g., hydrophobic toxins and pollutants) to the organic solvents must be given, since this may hinder the direct use of MCC-enriched solvents. These hydrophobic unwanted compounds might come from the organic residues themselves [57] or be produced by pathogens potentially enriched in the microbiomes. Thus, feedstock selection could be done accordingly to the final application. It is worth mentioning that vegetable oil components can inhibit many known pathogens [39].
A high unsaturated LCC content can promote immune responses in cattle and accumulation of conjugated linoleic acids (CLA) in animal-derived food products [25]. CLA are isomers of linoleic acid considered to have positive physiological effects on human health and are mostly sourced from dairy and meat products [59]. Thus, finding alternative ways to produce CLA could increase their dietary accessibility for human consumption. Microbial CLA production has been reported for different probiotic bacteria [60] and M. elsdenii [61], a chain-elongating microorganism. Although CLA were not detected in MCC-enriched sunflower oil, vegetable oils enriched with both CLA and MCC could be produced via chain elongation processes for feed or food purposes following pertinent regulations. MCC display differential effects on human health compared to unsaturated LCC [24] and CLA-rich food products are under development [62]. One related alternative of solvent choice is using oleyl alcohol, which is approved as indirect food additive by the FDA and regulates LCC uptake in mammals [63]. Biobased alkyl alcohols (e.g., oleyl alcohol) can be obtained from hydrogenation of plant-derived or microbially produced LCC.
The viscosities of 12 vegetable oils were experimentally determined as a function of temperature (5 to 95C) by means of a temperature-controlled rheometer. Viscosities of the oil samples decreased exponentially with temperature. Of the three models [modified Williams-Landel-Ferry (WLF), power law and Arrhenius] that were used to describe the effects of temperature on viscosity, the modified WLF model gave the best fit. The amounts of monounsaturated FA or polyunsaturated fatty acids (PUFA) highly correlated (R 2>0.82) with the viscosities of the oil samples whereas and the amounts of saturated or unsaturated FA. An exponential equation was therefore used to relate the viscosity of these vegetable oil samples to the amounts of monounsaturated FA or PUFA. The models developed are valuable for designing or evaluating systems and equipment that are involved in the storage, handling, and processing of vegetable oils.
The influence of FA ester chemical structures on the rheology and crystallization temperature of those compounds was evaluated using methyl, n-butyl, n-octyl, and 2-ethyl-1-hexyl FA esters with different chain lengths and different degrees of unsaturation. The rheological properties were analyzed in a high-precision rheometer at various temperatures, and the crystallization temperatures were determined by DSC. Esters produced from the esterification of pure FA and from the transesterification of vegetable oils (i.e., soybean, corn, linseed, and babassu coconut oils) were evaluated. The length of the FA chain was shown to have a marked influence on the viscosity and crystallization temperature of the systems, whereas branching affected only the crystallization temperature to a significant extent. The viscosity and crystallization temperature of the systems were also influenced by the degree of unsaturation. One double bond was shown to increase viscosity, whereas two or three double bonds caused a decrease in the viscosity of the systems. Unsaturation lowered the crystallization temperature in all cases, regardless of the number of double bonds. From all the oils studied, methyl esters from babassu coconut oil presented the lowest crystallization temperatures.
Another mechanism through which endotoxin can enter the circulation is through micelles. Since the endotoxin side chains are made up of fatty acids, endotoxins can be incorporated into the micelles and transported into the intestinal epithelial cell [54]. In intestinal epithelial cells, chylomicrons transport the absorbed lipids into various parts of the body. High fat administration has been shown to proportionately increase the endotoxin content of the chylomicron indicating that high fat consumption indeed enhances higher endotoxin transport into the intestinal epithelial cell and incorporation into chylomicron [11, 28]. Furthermore, even though the mechanism is not clear, high intake of fat has been shown to cause internalization of tight junction proteins and increase in the paracellular permeability to macro molecules including endotoxin [30]. Even though, this mode of endotoxin transport cannot be ruled out, we speculate that the rate of incorporation of fatty acids into micelles would not vary due to oil composition. Therefore, we propose that the difference in intestinal endotoxin transport we observed is primarily transcellular transport that involves lipid rafts and receptor mediated endocytosis [42]. 2ff7e9595c
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