Sustainable production of lipids from cocoa fatty acid distillate fermentation driven by adaptive evolution in Yarrowia lipolytica.

Single cell oil production using oleaginous yeasts is a promising alternative to animal and plant-derived lipids. But substrate costs for microbial fermentation are a major bottleneck. Using side streams as alternative to substrates like glucose, for growing yeast, is a potential cost-effective solution. By combining a previously reported process of growing yeasts on a solid cocoa fatty acid distillate side stream with adaptive evolution techniques, the growth of oleaginous yeast Yarrowia lipolytica was improved by 2-fold. The lipid titre was also boosted by more than 3-fold. Using transcriptomics, key genes were identified that are possibly involved in tailoring of lipid composition, side stream utilisation and enhancement of lipid titres. Candidate genes were also identified that might enable efficient growth and utilization of fatty acids and triacylglycerides found in cocoa fatty acid distillate. In summary, this research has improved the understanding of side stream utilisation for lipid production in oleaginous yeast.

Single cell protein and oil production from solid cocoa fatty acid distillates co-fed ethanol.

The use of solid lipid sidestreams have been overlooked as a feedstock for the production of microbial biomass for food and feed applications and little to no recent work has examined the utilization of solid fatty acid distillates (FADs), which are a significant residue from vegetable oil processing. Yarrowia lipolytica and Rhodosporidium toruloides cultivated on cocoa fatty acid distillates (CFAD) generated final cell dry weight values > 40 g/L, with strong productivity (3.3 g/L·h) and rich protein (>45%) and lipid content (>25%). Interestingly, microbial oils were > 65% unsaturated fatty acids, compared < 20% unsaturated content in FAD. Importantly, to overcome mass-transfer limitations associated with bioconversion of solid lipid residues, ethanol was applied as a co-substrate to solubilize FAD residues. Here, FAD residues from cocoa deodorization have been demonstrated to be high energy feedstocks that represent an attractive substrate for the production of both single cell protein and oil (SCPO).

Co-culture of Kluyveromyces marxianus and Meyerozyma guilliermondii with In Situ Product Recovery of 2-Phenylethanol.

Production of 2-phenylethanol (2-PE) via Kluyveromyces marxianus is well-established. However, co-culture with other microbes in combination with in situ product recovery (ISPR) yields improved selectivity and volumetric productivity. Fermentation ofK. marxianus (MUCL 53775) with direct inclusion of absorptive polymer Hytrel3548 achieved ISPR, but accumulation of the byproduct phenylethyl acetate (PEA) was strongly favored. Co-culture of K. marxianus (MUCL 53775) with Meyerozyma guilliermondii (MUCL 28072) with ISPR limited PEA production, thereby improving the 2-PE selectivity from 13 to 90%, compared to a pure culture of K. marxianus (MUCL 53775) under similar conditions. This improved the volumetric productivity by 85% compared to 2-PE ISPR with a pure culture of K. marxianus. This is the first report of co-culture in a two-phase fermentation for 2-PE bioproduction and demonstrates that interactions between co-culture and ISPR techniques can modulate bioproduction between 2-PE and byproduct PEA, and this technique will be explored for other strain combinations and for other high-value molecules of interest.

A newly characterized exopolysaccharide from Sanghuangporus sanghuang.

Sanghuangporus sanghuang is a well-known pharmacodynamic and economically important edible fungus associated with mulberry (Morus spp.). A distinctly new exopolysaccharide (EPS), designated SHP-2 was obtained from S. sanghuang P0988 broth, and its structure and anti-aging prosperity were characterized. SHP-2 was found to be composed of a back-bone of →4)-ß-Manp-(1→4)-α-Araf-(1→3,4)-α-Glcp(1→3,4)-α-Glcp-(1→3,4)-α-Glcp-(1→3,4)-α-Glcp-(1→3,4)-α-Glcp-(1→6)-α-Galp-(1→4)-ß-Manp-(1→ and five branches, including four α-D-Glcp-(1→ and one α-D-Manp-(1→SHP-2 was shown to increase antioxidant enzyme activities including catalase (CAT) and superoxide dismutase (SOD) activities, as well as trolox equivalent antioxidant (TEAC) capacity in serum of mice pre-treated with D-Gal, while reducing lipofuscin levels. SHP-2 exerted a favorable influence on immune organ coefficients and ameliorated the histopathological hepatic lesions and apoptosis in hepatocytes of D-galactose-aged mice almost in a dose-dependent manner. Using the same analytical methods, on comparison with previously studied EPS compounds (i.e. SHP-1), SHP-2 was found to have more complex structure, larger molecule weight, and different anti-aging properties. The results presented here suggest that not only does EPS bioactivity vary with respect to molecular structures and molecule weight, but that multiple structures with different activity can be expressed by a single fungal strain. These results may help understanding the anti-aging prosperity of these polysaccharides for use in health foods or dietary supplements.

Structural characterization of two endopolysaccharides from Phellinus sp. and their immunologic effects by intragastric administration in a healthy mammalian model.

Two purified endopolysaccharides derived from cultured Phellinus sp., individually named SHIP-1 and 2, were structurally characterized, along with an evaluation of their in vivo influential immunomodulatory activity in a healthy mammalian model. The structure of SHIP-1 was mainly composed of →4)-α-d-Fucp-(1→, →3,6)-α-d-Araf-(1→ and →2,4)-ß-d-Galp-(→, with four residuals of α-d-Manp-(1→ and one α-d-Glcp-(1→ as sidegroups, while the planar structure and the heteronuclear multiple-bond correlation of SHIP-2 were not able to be analyzed. Biochemical analysis in the healthy mice model demonstrated that SHIP-1 increased the concentrations of the detected cytokines in a dosage-dependent manner but not in a time-dependent way. SHIP-2 exerted a positive effect in a dose-dependent manner over time for interferon gamma (IFN-γ) and interleukin (IL)-2 cytokine production at elevated dosages of 200, or 350 mg kg-1 d-1, while IFN-alpha(α) and IL-4 production was observed only in a dosage-dependent manner even at high dosages. Thus, SHIP-1 and 2 significantly improved the immune response through the intragastric administration of the tested high dosages by increasing the production of cytokines in the healthy mice, and these polysaccharides could possibly be used as an immunopotentiator in health foods or dietary supplements.

Structural characterization and anti-aging activity of a novel extracellular polysaccharide from fungus Phellinus sp. in a mammalian system.

Little is known about the chemical structure of purified extracellular polysaccharides from Phellinus sp., a fungal species with known medicinal properties. A combination of IR spectroscopy, methylation analysis and NMR were performed for the structural analysis of a purified extracellular polysaccharide derived from Phellinus sp. culture, denoted as SHP-1, along with an evaluation of the anti-aging effect in vivo of the polysaccharide supplementation. The structure of SHP-1 was established, with a backbone composed of →2,4)-α-d-glucopyranose-(1→ and →2)-ß-d-mannopyranose-(1→ and two terminal glucopyranose branches. Biochemical analysis from mammalian animal experiments demonstrated that SHP-1 possesses the ability to enhance antioxidant enzyme activities, such as catalase (CAT) and superoxide dismutase (SOD) activities, Trolox equivalent antioxidant capacity (TEAC) in serum of d-galactose-aged mice, while reducing lipofuscin levels, another indicator of cell aging, indicating a potential association with anti-aging activities in a dose dependent manner. This compound had a favourable influence on immune organ indices, and a marked amelioration ability of histopathological hepatic lesions such as necrosis, karyolysis and reduced inflammation and apoptosis in mouse hepatocytes. These results suggest that SHP-1 has strong antioxidant activities and a significant protective effect against oxidative stress or hepatotoxicity induced by d-galactose in mice and it could be developed as a food ingredient or a pharmaceutical to prevent many age-associated diseases such as major depressive disorder and hepatotoxicity. To our knowledge, this is the first report on the antioxidant effects of a novel purified exopolysaccharide derived from Phellinus sp.

Heavy metals species affect fungal-bacterial synergism during the bioremediation of fluoranthene.

The co-occurrence of polycyclic aromatic hydrocarbons (PAHs) with heavy metals (HMs) is very common in contaminated soils, but the influence of HMs on fungal-bacterial synergism during PAH bioremediation has not been investigated. The bioremediation of fluoranthene-contaminated sand using co-cultures of Acremonium sp. P0997 and Bacillus subtilis showed increases of 109.4 and 9.8 % in degradation compared to pure bacterial and fungal cultures, respectively, removing 64.1 ± 1.4 % fluoanthene in total. The presence of Cu(2+) reduced fluoranthene removal to 53.7 ± 1.7 %, while inhibiting bacterial growth, and reducing translocation of bacteria on fungal hyphae by 49.5 %, in terms of the bacterial translocation ratio. Cu(2+) reduced bacterial diffusion by 46.8 and 31.9 %, as reflected by D (a bulk random motility diffusional coefficient) and D eff (the effective one-dimensional diffusion coefficient) compared to the control without HM supplementation, respectively. However, Mn(2+) resulted in a 78.2 ± 1.9 % fluoranthene degradation, representing an increase of 21.9 %, while enhancing bacterial growth and bacterial translocation on fungal hyphae, showing a 12.0 % increase in translocation ratio, with no observable impact on D and D eff. Hence, the presence of HMs has been shown to affect fungal-bacterial synergism in PAH degradation, and this effect differs with HM species.

The influence of naphthaleneacetic acid (NAA) and coumarin on flavonoid production by fungus Phellinus sp.: modeling of production kinetic profiles.

For the purpose of improving the fungal production of flavonoids, the influence of naphthaleneacetic acid (NAA) and coumarin on flavonoid production by fungus Phellinus sp. P0988 was investigated by developing the corresponding kinetics of flavonoid production in a 7-L bioreactor. Phellinus sp. was confirmed to form flavonoids in pellets and broth when cultivated in basic medium, and the optimum concentration of NAA and coumarin in medium for flavonoid production were determined to be 0.03 and 0.02 g/L, respectively. The developed unstructured mathematical models were in good agreement with the experimental results with respect to flavonoid production kinetic profiles with NAA and coumarin supplementation at optimum levels and revealed significant accuracy in terms of statistical consistency and robustness. Analysis of these kinetic processes indicated that NAA and coumarin supplementations imposed a stronger positive influence on flavonoid production and substrate consumption compared to their effects on cell growth. The separate addition of NAA and coumarin resulted in enhancements in final product accumulation and productivity, achieving final flavonoid concentrations of 3.60 and 2.75 g/L, respectively, and glucose consumption showed a significant decrease compared to the non-supplemented control as well. Also, the separate presence of NAA and coumarin respectively decreased maintenance coefficients (M s) from 2.48 in the control to 1.39 and 0.22, representing decreases of 43.9 and 91.1 %, respectively. The current study is the first known application of mathematical kinetic models to explore the influence of medium components adding on flavonoid production by fungi.

Bioaugmentation of soil contaminated with high-level crude oil through inoculation with mixed cultures including Acremonium sp.

Heavy contamination of soil with crude oil has caused significant negative environmental impacts and presents substantial hazards to human health. To explore a highly efficient bioaugmentation strategy for these contaminations, experiments were conducted over 180 days in soil heavily contaminated with crude oil (50,000 mg kg(-1)), with four treatments comprised of Bacillus subtilis inoculation with no further inoculation (I), or reinoculation after 100 days with either B. subtilis (II), Acremonium sp.(III), or a mixture of both organisms (IV). The removal values of total petroleum hydrocarbons were 60.1 ± 2.0, 60.05 ± 3.0, 71.3 ± 5.2 and 74.2 ± 2.7 % for treatment (I-IV), respectively. Treatments (III-IV) significantly enhanced the soil bioremediation compared with treatments (I-II) (p < 0.05). Furthermore, significantly (p < 0.05) greater rates of degradation for petroleum hydrocarbon fractions were observed in treatments (III-IV) compared to treatments (I-II), and this was especially the case with the degradative rates for polycyclic aromatic hydrocarbons and crude oil heavy fractions. Dehydrogenase activity in treatment (III-IV) containing Acremonium sp. showed a constant increase until the end of experiments. Therefore reinoculation with pure fungus or fungal-bacterial consortium should be considered as an effective strategy in bioaugmentation for soil heavily contaminated with crude oil.

Enhancing exopolysaccharide antioxidant formation and yield from Phellinus species through medium optimization studies.

The study was conducted to enhance exopolysaccharide (EPS) antioxidant formation (as measured by Trolox-Equivalent Antioxidant Capacity, TEAC) by Phellinus sp. P0988 through medium studies, while jointly increasing EPS yield. Out of seven medium components, four significant variables were identified based on linear models with statistical significance (p<0.05), namely rutin, FeSO4, and aspartate, which influenced TEAC, and malt extract, which influenced yield. Based on regression analysis of the experimental results from central composite design in response surface models, two second-order polynomial models adequately representing the relationship between these variables and EPS TEAC and yield were obtained, and the optimum concentration of these variables were determined and validated. Variable combinations for high EPS TEAC and yield were optimized through a desirability function test. The maximum EPS TEAC and yield reached 8.49 ± 0.32 mM and 10.92 ± 0.68 g/L, respectively, in a 7 L bioreactor under the optimum variable combinations.