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 bio-based 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

Candida freyschussii: an oleaginous yeast producing lipids from glycerol

A surplus of glycerol is obtained from biodiesel manufacturing and represents a waste product whose applications are lacking. Thus, the use of glycerol as substrate for fermentation processes yielding valuable products is very attractive. In this study, the utilization of glycerol as a growth substrate for the cultivation of oleaginous yeasts was explored with the aim to produce microbial oils. Forty strains of environmental non-conventional yeasts belonging to 19 different species were screened for the ability to grow on glycerol and produce intracellular lipids in a medium containing an excess of this carbon source (C:N = 48:1). Three strains, belonging to the species Candida freyschussii, Pichia farinosa, and Saccharomyces spencerorum, depleted 40 g/L glycerol within 120 h and produced intracellular lipids. C. freyschussii yielded the highest amounts, lipids accounting for the 33 % of biomass on dry basis. 1H-NMR analysis revealed that the lipid extract did not contain detectable free fatty acids and was composed mostly of triacylglycerols. Lipid composition, determined by GC-MS analysis, was similar to plant oils, and may be optimal feedstock for biodiesel production, being dominated by monounsaturated C16 and C18. As in other oleaginous yeasts, lipid production by C. freyschussii sp. increased with the increase of the C:N ratio of the medium, but growth was inhibited at glycerol concentrations higher than 40 g/L. As a result, lipid production was the highest with 40 g/L glycerol, yielding 4.7 g/L lipids, with a mean volumetric productivity of 0.15 g/L/h. In order to prevent growth inhibition over 40 g/L glycerol and extend the lipogenic phase, different fed-batch strategies were tried. The best performing processes took advantage from the feeding with concentrated media exhibiting the same C:N ratio of the basal medium, leading to very productive high cell density cultures. With the continuous feeding of 20X-concentrated medium, 29 g/L lipids (i.e. the 32 % of biomass) were obtained in 100 h of cultivation, with a mean volumetric productivity of 0.30 g/L/h. The values herein reported are among the highest yield and productivity values ever obtained for fermentative processes exploiting oleaginous fungi to produce lipids from glycerol. Therefore, C. freyschussii could be considered as an interesting microorganism to convert glycerol into microbial oils for biofuel industr

Microbiota Survey of Sliced Cooked Ham During the Secondary Shelf Life

Sliced cooked ham packaged in a modified atmosphere is a popular ready-to-eat product, subjected to abundant microbial contamination throughout its shelf life that can lead to deterioration of both sensorial properties and safety. In this study, the microbial load and the chemical–physical features of cooked ham of five producers were monitored for a period of 12 days after the opening of the packages (i.e., the secondary shelf life), during which the products were stored in a domestic refrigerator at 5.2 ± 0.6°C. The sensorial properties presented a perceivable decay after 8 days and became unacceptable after 12 days. High-performance liquid chromatography analysis and solid-phase microextraction coupled with gas chromatography profiling of volatile metabolites indicated that lactic acid, ethanol, acetic acid, acetoin, 3-methyl-1-butanol, and 2-3 butanediol were the main metabolites that characterized the evolution of the analyzed cooked ham. The microbiota was monitored by 16S ribosomal RNA gene profiling and culture-dependent techniques. Already at the opening of packages, all the products presented high microbial load, generally dominated by lactic acid bacteria, with evident differences among the products. The increase of lactic acid bacteria somehow protected samples from abundant contamination by other bacteria, concurring with the evolution of more safe products. This role was exerted by numerous Latilactobacillus, Leuconostoc, and Carnobacterium species, among which the most frequently detected were Latilactobacillus sakei, Latilactobacillus sakei carnosum, Leuconostoc mesenteroides, and Carnobacterium divergens. Some products presented more complex communities that encompassed Proteobacteria such as Moellerella wisconsensis, Proteus hauseri, Brochothrix thermosphacta, and less frequently Pseudomonas, Erwinia, and Massilia. Opportunistic pathogenic bacteria such as Escherichia coli and Vibrio sp. were found in small quantities. The yeasts Kazachstania servazzii and Debaryomyces hansenii occurred already at 0 days, whereas various species of Candida (Candida zeylanoides, Candida sake, Candida norvegica, and Candida glaebosa) were abundant only after 12 days. These results indicated that the microbiological contaminants overgrowing during the secondary shelf life did not derive from environmental cross-contamination at the opening of the tray but were already present when the packages were opened, highlighting the phases of production up to the packaging as those crucial in managing the safety risk associated to this product

Getting lipids from glycerol: new perspectives on biotechnological exploitation of Candida freyschussii

BACKGROUND: Microbial lipids represent a valuable alternative feedstock for biodiesel production when oleaginous microbes are cultured with inexpensive substrates in processes exhibiting high yield and productivity. In this perspective, crude glycerol is among the most promising raw materials for lipid production, because it is the costless residual of biodiesel production. Thus, cultivation of oleaginous yeasts in glycerol-based media is attracting great interest and natural biodiversity is increasingly explored to identify novel oleaginous species recycling this carbon source for growth and lipid production. RESULTS: Thirty-three yeasts strains belonging to 19 species were screened for the ability to grow and produce intracellular lipids in a pure glycerol-based medium with high C/N ratio. A minority of them consumed most of the glycerol and generated visible lipid bodies. Among them, Candida freyschussii ATCC 18737 was selected, because it exhibited the highest lipid production and glycerol conversion yield. Lipid production in this strain was positively affected by the increase of C/N ratio, but growth was inhibited by glycerol concentration higher than 40 g/L. In batch cultures, the highest lipid production (4.6 g/L), lipid content of biomass (33% w/w), and lipid volumetric productivity (0.15 g/L/h) were obtained with 40 g/L glycerol, during the course of a 30-h process. Fed-batch cultivation succeeded in preventing substrate inhibition and in achieving a high cell-density culture. The improved lipid production and volumetric productivity reached the remarkable high level of 28 g/L and 0.28 g/L/h, respectively. The lipids accumulated by C. freyschussii ATCC 18737 have similar fatty acid composition of plant oil indicating their potential use as biodiesel feedstock. Calculated physicochemical properties of a biodiesel produced with the lipids from C. freyschussii ATCC 18737 are expected to meet the European and American standards, being equal to those of rapeseed and palm biodiesel. CONCLUSIONS: C. freyschussii ATCC 18737 could be considered an interesting microorganism for utilization in biofuel industry. Cultivation of this yeast in media containing crude glycerol should be investigated deeper in order to evaluate whether it may find application in the valorization of the waste of biodiesel manufacturing

Indole and p-cresol in feces of healthy subjects: Concentration, kinetics, and correlation with microbiome

Indole and p-cresol are precursors of the most important uremic toxins, generated from the fermentation of amino acids tryptophan and tyrosine by the proteolytic community of intestinal bacteria. The present study focused on the relationship between the microbiome composition, the fecal levels of indole and p-cresol, and their kinetics of generation/degradation in fecal cultures. The concentration of indole and p-cresol, the volatilome, the dry weight, and the amount of ammonium and carbohydrates were analyzed in the feces of 10 healthy adults. Indole and p-cresol widely differed among samples, laying in the range of 1.0–19.5 μg/g and 1.2–173.4 μg/g, respectively. Higher fecal levels of indole and p-cresol were associated with lower carbohydrates and higher ammonium levels, that are markers of a more pronounced intestinal proteolytic metabolism. Positive relationship was observed also with the dry/wet weight ratio, indicator of prolonged intestinal retention of feces. p-cresol and indole presented a statistically significant negative correlation with OTUs of uncultured Bacteroidetes and Firmicutes, the former belonging to Bacteroides and the latter to the families Butyricicoccaceae (genus Butyricicoccus), Monoglobaceae (genus Monoglobus), Lachnospiraceae (genera Faecalibacterium, Roseburia, and Eubacterium ventriosum group). The kinetics of formation and/or degradation of indole and p-cresol was investigated in fecal slurries, supplemented with the precursor amino acids tryptophan and tyrosine in strict anaerobiosis. The presence of the precursors bursted indole production but had a lower effect on the rate of p-cresol formation. On the other hand, supplementation with indole reduced the net rate of formation. The taxa that positively correlated with fecal levels of uremic toxins presented a positive correlation also with p-cresol generation rate in biotransformation experiments. Moreover other bacterial groups were positively correlated with generation rate of p-cresol and indole, further expanding the range of taxa associated to production of p-cresol (Bacteroides, Alistipes, Eubacterium xylanophylum, and Barnesiella) and indole (e.g., Bacteroides, Ruminococcus torques, Balutia, Dialister, Butyricicoccus). The information herein presented contributes to disclose the relationships between microbiota composition and the production of uremic toxins, that could provide the basis for probiotic intervention on the gut microbiota, aimed to prevent the onset, hamper the progression, and alleviate the impact of nephropaties

Thermal adaptability of Kluyveromyces marxianus in recombinant protein production

Background: Kluyveromyces marxianus combines the ease of genetic manipulation and fermentation with the ability to efficiently secrete high molecular weight proteins, performing eukaryotic post-translational modifications. It is able to grow efficiently in a wide range of temperatures. The secretion performances were analyzed in the host K. marxianus L3 in the range between 5\ub0C and 40\ub0C by means of 3 different reporter proteins, since temperature appears a key parameter for production and secretion of recombinant proteins.Results: The recombinant strains were able to grow up to 40\ub0C and, along the tested temperature interval (5-40\ub0C), the specific growth rates (\u3bc) were generally lower as compared to those of the untransformed strain. Biomass yields were slightly affected by temperature, with the highest values reached at 15\ub0C and 30\ub0C. The secretion of the endogenous \u3b2-fructofuranosidase, used as an internal control, was efficient in the range of the tested temperature, as evaluated by assaying the enzyme activity in the culture supernatants. The endogenous \u3b2-fructofuranosidase production was temperature dependent, with the highest yield at 30\ub0C. The heterologous proteins HSA, GAA and Sod1p were all successfully produced and secreted between 5\ub0C and 40\ub0C, albeit each one presented a different optimal production temperature (15, 40, 5-30\ub0C for HSA, GAA and Sod1p, respectively).Conclusions: K. marxianus L3 has been identified as a promising and flexible cell factory. In a sole host, the optimization of growth temperatures for the efficient secretion of each individual protein can be carried out over a wide range of temperatures

Mining metagenomic whole genome sequences revealed subdominant but constant Lactobacillus population in the human gut microbiota

The genus Lactobacillus includes over 215 species that colonize plants, foods, sewage and the gastrointestinal tract (GIT) of humans and animals. In the GIT, Lactobacillus population can be made by true inhabitants or by bacteria occasionally ingested with fermented or spoiled foods, or with probiotics. This study longitudinally surveyed Lactobacillus species and strains in the feces of a healthy subject through whole genome sequencing (WGS) data-mining, in order to identify members of the permanent or transient populations. In three time-points (0, 670 and 700 d), 58 different species were identified, 16 of them being retrieved for the first time in human feces. L. rhamnosus, L. ruminis, L. delbrueckii, L. plantarum, L. casei and L. acidophilus were the most represented, with estimated amounts ranging between 6 and 8 Log (cells g-1), while the other were detected at 4 or 5 Log (cells g-1). 86 Lactobacillus strains belonging to 52 species were identified. 43 seemingly occupied the GIT as true residents, since were detected in a time span of almost 2 years in all the three samples or in 2 samples separated by 670 or 700 d. As a whole, a stable community of lactobacilli was disclosed, with wide and understudied biodiversity

Isolation of carotenoid-producing yeasts from an alpine glacier

Cold-adapted yeasts are increasingly being isolated from glacial environments, including Artic, Antarctic, and mountain glaciers. Psychrophilic yeast isolates mostly belong to Basidiomycota phylum, such as Cryptococcus, Mrakia, and Rhodotorula, and represent an understudied source of biodiversity for potential biotechnological applications. Since some basidiomycetous yeast genera (e.g. Rhodotorula, Phaffia, etc.) were demonstrated to produce commercially important carotenoids (e.g. β-carotene, torulene, torularhodin and astaxanthin), the present study aimed to obtain psychrophilic yeast isolates from the surface ice of two Italian glaciers to identify new pigment-producers. 23 yeast isolates were obtained. Among them, three isolates giving pigmented colonies was subjected to ITS1/ITS2 sequencing and were attributed to the Basidiomycetous yeasts Dioszegia sp., hodotorula mucilaginosa, and Rhodotorula laryngis. The strains were cultured batchwise in a carbon-rich medium at 15°C until the stationary phase was reached, then the pigments were extracted from freeze-dried biomass using DMSO:acetone mixture. Visible spectrum and HPLC-DAD analysis revealed the presence of carotenoid pigments. In batch cultures of Dioszegia sp., carotenoid production was growth-associated and yielded up to 3.4 mg/L of a molecule exhibiting an m/z ratio (568) consistent with the molecular weight of xanthophylls bearing 2 OH groups

Kinetics and metabolism of Bifidobacterium adolescentis MB 239 growing on glucose, galactose, lactose, and galactooligosaccharides

The kinetics and the metabolism of Bifidobacterium adolescentis MB 239 growing on galactooligosaccharides (GOS), lactose, galactose, and glucose were investigated. An unstructured unsegregated model for growth in batch cultures was developed, and kinetic parameters were calculated with a recursive algorithm. The growth rate and cellular yield were highest on galactose, followed by lactose and GOS, and were lowest on glucose. Lactate, acetate, and ethanol yields allowed the calculation of carbon fluxes toward fermentation products. Distributions between two- and three-carbon products were similar on all the carbohydrates (55 and 45%, respectively), but ethanol yields were different on glucose, GOS, lactose, and galactose, in decreasing order of production. Based on the stoichiometry of the fructose-6-phosphate shunt and on the carbon distribution among the products, the ATP yield was calculated. The highest yield was obtained on galactose, while the yields were 5, 8, and 25% lower on lactose, GOS, and glucose, respectively. Therefore, a correspondence among ethanol production, low ATP yields, and low biomass production was established, demonstrating that carbohydrate preferences may result from different distributions of carbon fluxes through the fermentative pathway. During the fermentation of a GOS mixture, substrate selectivity based on the degree of polymerization was exhibited, since lactose and the trisaccharide were the first to be consumed, while a delay was observed until longer oligosaccharides were utilized. Throughout the growth on both lactose and GOS, galactose accumulated in the cultural broth, suggesting that beta(1-4) galactosides can be hydrolyzed before they are taken up

Fermentative processes for the upcycling of xylose to xylitol by immobilized cells of Pichia fermentans WC1507

Xylitol is a pentose-polyol widely applied in the food and pharmaceutical industry. It can be produced from lignocellulosic biomass, valorizing second-generation feedstocks. Biotechnological production of xylitol requires scalable solutions suitable for industrial scale processes. Immobilized-cells systems offer numerous advantages. Although fungal pellet carriers have gained attention, their application in xylitol production remains unexplored. In this study, the yeast strain P. fermentans WC 1507 was employed for xylitol production. The optimal conditions were observed with free-cell cultures at pH above 3.5, low oxygenation, and medium containing (NH4)2SO4 and yeast extract as nitrogen sources (xylitol titer 79.4 g/L, YP/S 66.3%, and volumetric productivity 1.3 g/L/h). Yeast cells were immobilized using inactive Aspergillus oryzae pellet mycelial carrier (MC) and alginate beads (AB) and were tested in flasks over three consecutive production runs. Additionally, the effect of a 0.2% w/v alginate layer, coating the outer surface of the carriers (cMC and cAB, respectively), was examined. While YP/S values observed with both immobilized and free cells were similar, the immobilized cells exhibited lower final xylitol titer and volumetric productivity, likely due to mass transfer limitations. AB and cAB outperformed MC and cMC. The uncoated AB carriers were tested in a laboratory-scale airlift bioreactor, which demonstrated a progressive increase in xylitol production in a repeated batch process: in the third run, a xylitol titer of 63.0 g/L, YP/S of 61.5%, and volumetric productivity of 0.52 g/L/h were achieved. This study confirmed P. fermentans WC 1507 as a promising strain for xylitol production in both free- and entrapped-cells systems. Considering the performance of the wild strain, a metabolic engineering intervention aiming at further improving the efficiency of xylitol production could be justified. MC and AB proved to be viable supports for cell immobilization, but additional process development is necessary to identify the optimal bioreactor configuration and fermentation conditions