Alan Leonardi

Single cell oils of the cold-adapted oleaginous yeast Rhodotorula glacialis DBVPG 4785

BackgroundThe production of microbial lipids has attracted considerable interest during the past decade since they can be successfully used to produce biodiesel by catalyzed transesterification with short chain alcohols. Certain yeast species, including several psychrophilic isolates, are oleaginous and accumulate lipids from 20 to 70% of biomass under appropriate cultivation conditions. Among them, Rhodotorula glacialis is a psychrophilic basidiomycetous species capable to accumulate intracellular lipids.ResultsRhodotorula glacialis DBVPG 4785 is an oleaginous psychrophilic yeast isolated from a glacial environment. Despite its origin, the strain abundantly grew and accumulated lipids between -3 to 20°C. The temperature did not influence the yield coefficients of both biomass and lipids production, but had positive effect on the growth rate and thus on volumetric productivity of lipid. In glucose-based media, cellular multiplication occurred first, while the lipogenic phase followed whenever the culture was limited by a nutrient other than glucose. The extent of the carbon excess had positive effects on triacylglycerols production, that was maximum with 120 g L-1 glucose, in terms of lipid concentration (19 g L-1), lipid/biomass (68%) and lipid/glucose yields (16%). Both glucose concentration and growth temperature influenced the composition of fatty acids, whose unsaturation degree decreased when the temperature or glucose excess increased.ConclusionsThis study is the first proposed biotechnological application for Rhodotorula glacialis species, whose oleaginous biomass accumulates high amounts of lipids within a wide range of temperatures through appropriate cultivation C:N ratio. Although R. glacialis DBVPG 4785 is a cold adapted yeast, lipid production occurs over a broad range of temperatures and it can be considered an interesting microorganism for the production of single cell oils.

Bioconversion of soy isoflavones daidzin and daidzein by Bifidobacterium strains

Twenty-two strains of Bifidobacterium, representative of eight major species of human origin, were screened for their ability to transform the isoflavones daidzin and daidzein. Most of the strains released the aglycone from daidzin and 12 gave yields higher than 90%. The kinetics of growth, daidzin consumption, and daidzein production indicated that the hydrolytic activity occurred during the growth. The supernatant of the majority of the strains did not release the aglycone from daidzin, suggesting that cell-associated β-glucosidases (β-Glu) are mainly responsible for the metabolism of soybean glyco-conjugates. Cell-associated β-Glu was mainly intracellular and significantly varied among the species and the strains. The lack of β-Glu was correlated with the inability to hydrolyze daidzin. Although S-equol production by anaerobic intestinal bacteria has been established, information on S-equol-producing bifidobacteria is contradictory. In this study, 22 bifidobacteria failed to transform daidzein into reduced metabolites under all the experimental conditions, excluding any role in the reductive pathway of daidzein toward the production of S-equol. These results suggest that selected probiotic strains of Bifidobacterium can be used to speed up the release of daidzein, improving its bioavailability for absorption by colonic mucosa and/or biotransformation to S-equol by other intestinal microorganisms.

In vitro transformation of chlorogenic acid by human gut microbiota.

SCOPE Chlorogenic acid (3-O-caffeoyl-quinic acid, C-QA), the caffeic ester of quinic acid, is one of the most abundant phenolic acids in Western diet. The majority of C-QA escapes absorption in the small intestine and reaches the colon, where the resident microbiota transforms it into several metabolites. C-QA conversion by the gut microbiota from nine subjects was compared to evaluate the variability of bacterial metabolism. It was investigated whether a potentially probiotic Bifidobacterium strain, capable of C-QA hydrolysis, could affect C-QA fate. METHODS AND RESULTS Bioconversion experiments exploiting the microbiota from diverse subjects revealed that C-QA was metabolized through a succession of hydrogenation, dexydroxylation and ester hydrolysis, occurring in different order among the subjects. Transformation may proceed also through quinic acid residue breakdown, since caffeoyl-glycerol intermediates were identified (HPLC-MS/MS, Q-TOF). All the pathways converged on 3-(3-hydroxyphenyl)-propanoic acid, which was transformed to hydroxyphenyl-ethanol and/or phenylacetic acid in few subjects. A strain of Bifidobacterium animalis able to hydrolyze C-QA was added to microbiota cultures. It affected microbial composition but not to such an extent that C-QA metabolism was modified. CONCLUSION A picture of the variability of microbiota C-QA transformations among subjects is provided. The transformation route through caffeoyl-glycerol intermediates is described for the first time.

Getting Lipids for Biodiesel Production from Oleaginous Fungi

Biomass-based biofuel production represents a pivotal approach to face high energy prices and potential depletion of crude oils reservoirs, to reduce greenhouse gas emissions, and to enhance a sustainable economy (Zinoviev et al., 2010). Microbial lipids can represent a valuable alternative feedstock for biodiesel production, and a potential solution for a biobased economy. Nowadays, the production of biodiesel is based mostly on plant oils, even though animal fats, and algal oils can also be used. In particular, soybean, rapeseed, and palm oils are adopted as the major feedstock for biodiesel production. They are produced on agricultural land, opening the debate on the impact of the expansion of bioenergy crop cultures, which displace land from food production. Furthermore, their price restricts the large-scale development of biodiesel to some extent. In order to meet the increasing demand of biodiesel production, other oil sources have been explored. Recently, the development of processes to produce single cell oil (SCO) by using heterotrophic oleaginous microorganisms has triggered significant attention (Azocar et al., 2010). These organisms accumulate lipids, mostly consisting of triacylglycerols (TAG), that form the storage fraction of the cell. The occurrence of TAG as reserve compounds is widespread among all eukaryotic organisms such as fungi, plants and animals, whereas it has only rarely been described in bacteria (Meng et al., 2009). In fact, bacteria generally accumulate polyhydroxyalkanoates as storage compound and only few bacterial species, belonging to the actinobacterial genera Mycobacterium, Streptomyces, Rhodococcus and Nocardia produce relevant amounts of lipids (Alvarez & Steinbuchel, 2002). Among heterotrophic microorgansisms, oleaginous fungi, including both molds and yeasts, are increasingly been reported as good TAG producers. This chapter will focus on current knowledge advances in their metabolism, physiology, and in the result achieved in strain improvement, process engineering and raw material exploitation.

Hydrolysis of the Rutinose-Conjugates Flavonoids Rutin and Hesperidin by the Gut Microbiota and Bifidobacteria

Flavonols and flavanones are polyphenols exerting many healthy biological activities. They are often glycosylated by rutinose, which hampers absorption in the small intestine. Therefore they require the gut microbiota to release the aglycone and enable colonic absorption. The role of the gut microbiota and bifidobacteria in the release of the aglycones from two major rutinosides, hesperidin and rutin, was investigated. In bioconversion experiments, the microbiota removed rutinose from both rutin and hesperidin, even though complete hydrolysis was not obtained. To investigate whether bifidobacteria can participate to the hydrolysis of rutinosides, 33 strains were screened. Rutin was resistant to hydrolysis by all the strains. Among six tested species, mostly Bifidobacterium catenulatum and Bifidobacterium pseudocatenultum were able to hydrolyze hesperidin, by means of a cell-associated activity. This result is in agreement with the presence of a putative α-l-rhamnosidase in the genome of B. pseudocatenulatum, while most of the available genome sequences of bifidobacteria aside from this species do not bear this sequence. Even though B. pseudocatenulatum may contribute to the release of the aglycone from certain rutinose-conjugated polyphenols, such as hesperidin, it remains to be clarified whether this species may exert a role in affecting the bioavailability of the rutinoside in vivo.

Potential Impact of Probiotic Consumption on the Bioactivity of Dietary Phytochemicals

Many healthy phytochemicals occur in food in the form of esters, glycoconjugates, or polymers, which are not directly bioavailable. Probiotic lactobacilli and bifidobacteria, which have evolved within the colonic ecosystem where indigestible oligo- and polysaccharides are their sole carbon sources, bear several glycosyl-hydrolases and can contribute to release the aglycones from glycoconjugated phytochemicals. Among the glycosyl-hydrolases, β-glucosidases are the most pertinent, because many phytochemicals are glucoconjugates. β-Glucosidase-positive probiotic bacteria were proved to release the aglycones of isoflavones and lignans in vitro, but studies in vivo are scarce. A positive correlation between probiotic consumption and urinary and/or plasma levels of isoflavone or lignan metabolites was not established. However, the strains used in the trials were not validated for the enzymatic properties or for the ability to hydrolyze lignans or isoflavones. Thus, activation of specific phytochemicals by probiotic bacteria still needs substantial efforts to be proved.

1H NMR of native and azide-inhibited laccase from Rhus vernicifera

The 1H NMR spectra of the fully oxidized Rhus vernicifera laccase and of its 1:1 and 2:1 azide adducts are reported for the first time. These spectra, which are the first so far reported for a multi copper oxidase, contain a number of broad hyperfine-shifted resonances in the high frequency region of the spectrum, which are attributed to the metal binding residues of the mononuclear T1 center. The differences between the patterns of the hyperfine resonances of the free enzyme and its azide derivatives suggest that the alterations in the structural properties of the T3 site induced by the binding of the first azide molecule induce a limited alteration of the spin density distribution over the T1 copper ligands. Overall, these data demonstrate that 1H NMR can be fruitfully applied to characterize the electronic properties of the metal sites of blue oxidases at room temperature.

Conjugated Linoleic Acid Production by Bifidobacteria: Screening, Kinetic, and Composition.

Conjugated linoleic acids (CLA) are positional and geometric isomers of linoleic acid involved in a number of health aspects. In humans, CLA production is performed by gut microbiota, including some species of potential probiotic bifidobacteria. 128 strains of 31 Bifidobacterium species were screened with a spectrophotometric assay to identify novel CLA producers. Most species were nonproducers, while producers belonged to B. breve and B. pseudocatenulatum. GC-MS revealed that CLA producer strains yielded 9cis,11trans-CLA and 9trans,11trans-CLA, without any production of other isomers. Hydroxylated forms of LA were absent in producer strains, suggesting that the myosin-cross-reactive antigen (MCRA) protein that exerts hydratase activity is not involved in LA isomerization. Moreover, both CLA producer and nonproducer species bear a MCRA homologue. The strain B. breve WC 0421 was the best CLA producer, converting LA into 68.8% 9cis,11trans-CLA and 25.1% 9trans,11trans-CLA. Production occurred mostly during the lag and the exponential phase. For the first time, production and incorporation of CLA in biomass were assessed. B. breve WC 0421 stored CLA in the form of free fatty acids, without changing the composition of the esterified fatty acids, which mainly occurred in the plasmatic membrane.

Zinc Uptake by Lactic Acid Bacteria

The study aims to investigate zinc biosorption by strains of lactobacilli and bifidobacteria with a view to exploit them as organic matrixes for zinc dietary supplementation. Sixteen human strains of Lactobacillus and Bifidobacterium were assayed for zinc uptake. The minimum inhibitory concentration of zinc salts differed among the strains, but was never below 15 mmol L(-1). When cultured in MRS broth containing 10 mmol L(-1) ZnSO4, all the strains were capable of accumulating zinc in the range between 11 and 135 μmol g(-1). The highest amount of cell-bound zinc was obtained in L. acidophilus WC 0203. pH-controlled batch cultures of this strain revealed that zinc uptake started in the growth phase, but occurred mostly during the stationary phase. Pasteurized and viable cultures accumulated similar amount of zinc, suggesting that a nonmetabolically mediated mechanism is involved in zinc uptake. These results provide new perspectives on the specific use of probiotics, since L. acidophilus WC 0203 could function as an organic matrix for zinc incorporation. The bioavailability of Lactobacillus-bound zinc deserves to be investigated to provide a future basis for optimization of zinc supplementation or fortification.

Rapid method for screening enoate reductase activity in yeasts

Old Yellow Enzymes (OYEs, EC 1.6.99.1) are flavin-dependent oxidoreductases that catalyze the asymmetric reduction of electron-poor alkenes (enoate reductase activity). Since OYEs are involved in detoxification of acrolein, a high-throughput method for selecting yeasts expressing high enoate reductase activity, based on their acrolein resistance, was developed. The screening method was based on the measurement of growth in acrolein-supplemented medium, in 96-well microtiter plate cultures. A quantitative descriptor (Acrolein Resistance Factor=ARF) was firstly designed for quantifying the influence of both acrolein concentration and time of exposure on yeast growth. Besides, the efficiency of bioconversion of ketoisophorone (KIP) was exploited to measure OYE activity. In order to validate the method, ARF was correlated with the bioconversion of KIP on thirty yeast strains, belonging to 7 genera. With only a few exceptions, all strains exhibiting the highest ARF also displayed the maximum OYE activity. The presence of OYE genes in the strains showing OYE activity was confirmed by PCR amplification. Based on the results herein reported, this method should be profitably used as a fast screening procedure aimed at selecting outstanding strains for whole-cell bioconversions and could open many possibilities for the isolation and the biocatalytic exploitation of new OYEs from yeasts.