Traditional and new directions in biotechnological applications of yeast species of the genus Rhodotorula

Drożdże z rodzaju Rhodotorula występują powszechnie w środowisku. Do niedawna postrzegane były głównie jako psujące lub zanieczyszczające żywność saprofity. Jednak odkrycie przez naukowców wielu nowych możliwości ich wykorzystania pozwala sądzić, że mogą stać się w przyszłości znaczącą grupą drobnoustrojów przemysłowych. W niniejszym opracowaniu przedstawiono niektóre kierunki zastosowania drożdży z rodzaju Rhodotorula. Karotenoidy, egzopolisacharydy oraz enzymy produkowane przez biomasę komórkową tych drożdży mogą być wykorzystywane w wielu branżach przemysłu. Równie ważnym aspektem, uwzględnianym przy ocenie przydatności drożdży, jest wykorzystanie ich biomasy komórkowej w procesach wiązania jonów metali bądź biodegradacji odpadów.The yeast species of the genus Rhodotorula are ubiquitous in the human environment. Until recently, they have been mainly perceived as saprophytes, which spoil or contaminate food products. However, owing to many new options discovered by scientists of using them, they could be supposed to become a significant group of micro-organisms in the future. In this paper, some directions of using yeast of the genus Rhodotorula are presented. Carotenoids, exopolysaccharides, and enzymes produced by the biomass of Rhodotorula strain can be applied in many industrial fields. While assessing the usefulness of yeast, another more important aspect is considered, namely, the utilization of their cell biomass in the processes of binding metal ions or in biodegradation of waste/refuse

Symbioses between bacteria and gutless marine oligochaetes

The first description of a gutless marine oligochaete was published in 1977 (Jamieson 1977), but it was not until 1979 that the reduction of a mouth and gut in this species and several other marine tubificid worms was recognized (Erséus 1979a,b; Giere 1979). At the time, the only other free-living worms known to lack a mouth or gut were pogonophores (now called Frenulata) that were most commonly found buried deep in the reducing sediments of continental slopes. Extensive studies on these very long and thin pogonophore worms indicated that their high surface areas enabled them to gain their nutrition from the uptake of dissolved organic compounds from the environment (Southward and Southward 1980). It was therefore assumed that the gutless oligochaetes that are also quite thin (0.1–0.2 mm) and relatively long (up to 2–3 cm), also gain their nutrition through the diffusive uptake of organic compounds from the sediment pore waters

The influence of glycerol as a carbon source and ph of cultivation medium on biosynthesis of cell wall polymers of candida utilis and kluyveromyces fragilis yeasts

Celem badań było określenie możliwości wykorzystania glicerolu w hodowli drożdży Candida utilis oraz Kluyveromyces fragilis ukierunkowanej na biosyntezę β-glukanu i/lub mannoprotein tworzących strukturę ściany komórkowej tych grzybów. Preparaty ścian uzyskiwano na drodze autolizy komórek drożdży. Poddawano je następnie frakcjonowaniu w warunkach alkalicznych na poszczególne polisacharydy. Stwierdzono zróżnicowanie zawartości β(1,3)-/β(1,6)-glukanów i mannoprotein w ścianach badanych drożdży, zależne od szczepu, stężenia glicerolu oraz pH podłoży hodowlanych. Istotne zwiększenie zawartości cukrów ogółem i β(1,3)-/β(1,6)-glukanów w ścianie drożdży Candida utilis ATTC 9950 odnotowano po hodowli w podłożu o pH 4,0 zawierającym 2% glicerolu. W preparatach ścian omawianego szczepu stwierdzono wówczas około 75% cukrów ogółem i około 53% β(1,3)-/(1,6)-glukanu. Drożdże Kluyveromyces fragilis R11 okazały się lepszym źródłem mannoprotein. W preparatach komórek z podłoży o pH 7, zawierających 3 lub 5% glicerolu, odnotowano około 30,5% tych polimerów.The structure of yeast cell wall is mainly composed of polymers such as β-glucans and α-mannan (present as mannoprotein). It is well known that yeast β-glucans and mannoprotein exhibit a range of bioactive properties in humans and animals, like anti-toxic, anti-mutagenic, anticancerous and anti-oxidative activity, stimulation of immunological response as well as antibacterial properties. The content of β-glucans and mannoprotein in cell wall of unicellular fungi is connected to growth conditions. This work reports on the infl uence of glycerol as a carbon source and pH of the medium on structural cell wall polymers (β-glucan and mannoprotein) biosynthesis of Candida utilis ATTC 9950 and Kluyveromyces fragilis R11. The experimental cultivations of investigated yeast strains were performed in control YPD medium and in a model mediums where glucose was replaced with glycerol in the amount of 2, 3 and 5% (w/v). All mediums were prepared in three pH variants i.e. 4.0, 5.0 and 7.0. The preparations of cell walls of yeasts from the experimental cultivations were achieved via 24-h cell autolysis. The obtained cell wall preparations were subjected to fractionation on particular structural polysaccharides, i.e. β(1,3)-glucan, β(1,6)-glucan and mannoproteiny, by extraction in alkaline conditions. The content of reducing carbohydrates (as glucose) was analyzed using colorimetric method (λ = 540 nm) with 3,5-dinitrosalicylic acid after hydrolysis of particular fractions in acidic conditions. Results of the investigation demonstrated that cultivation of Candida utilis ATTC 9950 and Kluyveromyces fragilis R11 on glicerol, low-cost substrates from biodiesel production, may intensify the biosynthesis of cell wall polymers. The tendency depended on cultivation medium and strain. For yeast of genus Candida the portion of polysaccharides in total, as well as β(1,3)/(1,6)-glucan content were highest after cultivation on medium containing 2% of glycerol and pH 4.0. It was 75% and 53% respectively. Depending on growth conditions, the Candida utilis ATTC 9950 strain contained from 2 to 4 times more β(1,3)/(1,6)-glucan in cell wall structure comparing with Kluyveromyces fragilis yeasts. At the same time, Kluyveromyces fragilis R11 strain was more effi cient source of mannoprotein comparing with Candida yeast. Preparations of cell walls of Kluyveromyces fragilis after yeast cultivation in mediums with 3 and 5% of glycerol pH 7.0 contained app. 30.5% of mannoproteins. The results confi rmed that cultivation in medium with glycerol as a carbon source contributed to obtaining the biomass of Candida utilis and Kluyveromyces fragilis with increased portion of functional cell wall polymers. Further studies should be oriented towards an optimization of cultivation conditions for effi cient biosynthesis of β-glucans or mannoprotein and to establish their functional properties

Phylogeny of 16S rRNA, ribulose 1,5-bisphosphate carboxylase/oxygenase, and adenosine 5 '-phosphosulfate reductase genes from gamma- and alphaproteobacterial symbionts in gutless marine worms (Oligochaeta) from Bermuda and the Bahamas

Gutless oligochaetes are small marine worms that live in obligate associations with bacterial endosymbionts. While symbionts from several host species belonging to the genus Olavius have been described, little is known of the symbionts from the host genus Inanidrilus. In this study, the diversity of bacterial endosymbionts in Inanidrilus leukodermatus from Bermuda and Inanidrilus makropetalos from the Bahamas was investigated using comparative sequence analysis of the 16S rRNA gene and fluorescence in situ hybridization. As in all other gutless oligochaetes examined to date, I. leukodermatus and I. makropetalos harbor large, oval bacteria identified as Gamma 1 symbionts. The presence of genes coding for ribulose-1,5-bisphosphate carboxylase/oxygenase form I (cbbL) and adenosine 5′-phosphosulfate reductase (aprA) supports earlier studies indicating that these symbionts are chemoautotrophic sulfur oxidizers. Alphaproteobacteria, previously identified only in the gutless oligochaete Olavius loisae from the southwest Pacific Ocean, coexist with the Gamma 1 symbionts in both I. leukodermatus and I. makropetalos, with the former harboring four and the latter two alphaproteobacterial phylotypes. The presence of these symbionts in hosts from such geographically distant oceans as the Atlantic and Pacific suggests that symbioses with alphaproteobacterial symbionts may be widespread in gutless oligochaetes. The high phylogenetic diversity of bacterial endosymbionts in two species of the genus Inanidrilus, previously known only from members of the genus Olavius, shows that the stable coexistence of multiple symbionts is a common feature in gutless oligochaetes

Multiple bacterial symbionts in two species of co-occurring gutless oligochaete worms from Mediterranean sea grass sediments

Gutless oligochaete worms are found worldwide in the pore waters of marine sediments and live in symbiosis with chemoautotrophic sulfur-oxidizing bacteria. In the Mediterranean, two species of gutless oligochaete worms, Olavius algarvensis and O. ilvae, co-occur in sediments around sea grass beds. These sediments have extremely low sulfide concentrations (< 1 microM), raising the question if O. ilvae, as shown previously for O. algarvensis, also harbours sulfate-reducing symbionts that provide its sulfur-oxidizing symbionts with reduced sulfur compounds. In this study, we used fluorescence in situ hybridization (FISH) and comparative sequence analysis of genes for 16S rRNA, sulfur metabolism (aprA and dsrAB), and autotrophic carbon fixation (cbbL) to examine the microbial community of O. ilvae and re-examine the O. algarvensis symbiosis. In addition to the four previously described symbionts of O. algarvensis, in this study a fifth symbiont belonging to the Spirochaetes was found in these hosts. The symbiotic community of O. ilvae was similar to that of O. algarvensis and also included two gammaproteobacterial sulfur oxidizers and two deltaproteobacterial sulfate reducers, but not a spirochete. The phylogenetic and metabolic similarity of the symbiotic communities in these two co-occurring host species that are not closely related to each other indicates that syntrophic sulfur cycling provides a strong selective advantage to these worms in their sulfide-poor environment

Evaluation of the ability of the intraclellular fat biosynthesis by Rhodotorula gracilis yeast in media containing potato wastewater enriched with glycerol

Celem badań było określenie zdolności wzrostu drożdży Rhodotorula gracilis i biosyntezy przez nie wewnątrzkomórkowego tłuszczu (SCO – Single Cell Oil) podczas 72-godzinnej hodowli wgłębnej w podłożach zawierających ziemniaczaną odpadową wodę sokową wzbogaconą glicerolem w ilościach 5, 10, 15 i 20% obj. Największy plon biomasy komórkowej (28,65 gs.s.·dm⁻³) uzyskano w podłożu zawierającym 5% obj. glicerolu, a najwięcej wewnątrzkomórkowego tłuszczu (25,57 g·100 gs.s.⁻¹) stwierdzono w biomasie drożdży rosnących w podłożach zawierających 20% obj. glicerolu. Ta ilość tłuszczu była 3 i 5 razy większa w porównaniu z eksperymentami prowadzonymi w podłożu kontrolnym YPD (8,87 g·100 gs.s.⁻¹) i doświadczalnym bez gliceryny (4,81 g·100 gs.s.⁻¹). W podłożach wzbogaconych 15 lub 20% obj. dodatkiem glicerolu największa objętościowa produktywność biosyntezy tłuszczu w komórkach stosowanego szczepu drożdży przekraczała 5 g·dm⁻³. Hodowla wgłębna drożdży Rhodotorula gracilis w podłożu stanowiącym odpadową wodę sokową ziemniaczaną wzbogaconą glicerolem w ilości 15–20% obj. prowadzi do biosyntezy tłuszczu wewnątrzkomórkowego.The biosynthesis of intracellular fat by oleaginous yeast strains based on alternative sources of carbon, nitrogen, phosphorus and mineral ingredients can provide the opportunity for the utilization of the industrial wastes. The culture media for SCO production can include glycerol from biodiesel production as a source of carbon as well as potato wastewater as a source of nitrogen. Potato wastewater is a waste of the production of protein feed obtained by proteins thermal-acid coagulation from potato juice. The aim of the study was to determine the ability of Rhodotorula gracilis yeast to the growth and intracellular biosynthesis of fat during 72-hour bath cultivation in experimental media containing potato wastewater enriched with glycerol. Experimental media contained potato wastewater and diverse addition of glycerol in quantities of 5, 10, 15 or 20% vol. Control medium was YPD. Potato wastewater originated from PEPEES SA Food Industry in Łomża (Central East Poland). The biomass yield, intracellular fat content as well as glycerol in cultivation media content were determined during yeast cultivation. Biomass yield was performed by gravimetric method, while glycerol content by the Milchert’s method. The intracellular fat content was determined after extraction according to the Soxhlet method with petroleum ether. The highest yield of the cell biomass (28.65 gs.s.·dm⁻³) was obtained in the experimental medium with wastewater and 5% of glycerol. Culturing of the yeast of Rhodotorula gracilis in medium containing potato wastewater media enriched with 15 or 20% glycerol leads to the biosynthesis of intracellular fat. The intracellular biosynthesis of fat in the experimental media glycerol occurred in the stationary growth phase. The highest intracellular fat (25.57 g·100 gs.s.⁻¹) was found in yeast biomass from the experimental media containing 20% glycerol. In comparison to the fat content in the biomass from YPD control medium (8.87 g·100 gs.s.⁻¹) and experimental without glycerol (4.81 g·100 gs.s.⁻¹) the value was about 3 and 5 times greater respectively. The volumetric productivity of fat in cells of the yeast strain tested exceeded 5 g·dm⁻³ in potato wastewater media enriched with 15 or 20% glycerol. Increasing the glycerol content in the experimental media (from 5 to 20% vol) led to a reduction in glycerol utilization by yeast (from 86 to 59%). The fact of incomplete utilization of glycerol in experimental media should result in an extension of the submerged culture over 72 hours

An attempt to application glycerol and potato wastewater to production carotenoids by Rhodotorula gracilis yeast

Celem badań było określenie zdolności biosyntezy karotenoidów przez drożdże Rhodotorula gracilis podczas hodowli wgłębnych w podłożach zawierających ziemniaczaną wodę sokową oraz glicerol. Największy plon biomasy komórkowej (ponad 30 g ss·dm⁻³) stwierdzono w podłożach z dodatkiem 3 i 5% glicerolu. Po hodowli w podłożu z dodatkiem 3% glicerolu uzyskano najwyższy stopień wykorzystania glicerolu (85,7%) oraz białka (69,3%), a także znaczną redukcję wskaźnika ChZT (84,8%) podłoża. Wysokie stężenie związków stanowiących źródło węgla zahamowało biosyntezę karotenoidów przez badane drożdże, a ich zawartość w biomasie po hodowli w podłożach z glicerolem była ponad trzy razy mniejsza (34,6–40,9 μg·g⁻¹ss) w porównaniu do podłoża kontrolnego (142,6 μg·g⁻¹ss). Dominującym związkiem syntetyzowanym przez drożdże w podłożach z glicerolem był torulen, a jego udział stanowił 66,9–69,7% ogólnej zawartości karotenoidów. Na podstawie uzyskanych wyników stwierdzono, że badany szczep drożdży może stać się w przyszłości nowym źródłem karotenoidów, jednak z uwagi na mniejszą objętościową produktywność tych związków (0,9–1,23 mg·dm⁻³) konieczne są dalsze badania w celu zwiększenia wydajności ich biosyntezy w podłożach z ziemniaczaną wodą sokową i glicerolem.Carotenoids belong to the group of pigments widely used in different industries. Nowadays it is being looked for new methods of producing this compounds, and the use of microorganisms can constitute an new alternative for chemical synthesis. In order to reduce the costs of microbial synthesis, as components of culture media industrial wastes can be used. The aim of this study was to determine the ability of Rhodotorula gracilis yeast to biosynthesis of carotenoids during the cultivation in media with potato wastewater and glycerol. As a control medium not supplemented potato wastewater was used. Three experimental media were used for cultivation potato wastewater and glycerol in an amount of 3, 5 and 10 g·100 cm⁻³. Cultivation of yeast was carried out on a reciprocating shaker (200 rpm) for 96 h. Biomass yield was performed by weight method, while optical density by the spectrophotometric method. During the cultivation, glycerol (chemical method), reducing sugars (Miller method) and protein (Kjeldahl’s method) concentration in the experimental media and the chemical-oxygen demand indicator (dichromate method) of media were determined. Carotenoids content in the yeast biomass were determined by spectrophotometric method and their contribution by HPLC-UV. The highest yield of the cell biomass (more than 30 g d.m.·dm⁻³) was obtained in the experimental media with potato wastewater supplemented with a 3 and 5% addition of glycerol. After cultivation of R. gracilis yeast in medium with 3% addition of glycerol the highest degree of glycerol content (85.7%), total protein content (69.3%) and COD index (84.8%) reduction were observed. Base of this results, it was found that there is a possibility of simultaneous biodegradation of glycerol and potato wastewater during the cultivation of tested yeast strain. The highest carotenoids content in the yeast biomass (142.6 μg·g⁻¹ d.m.) was obtained in control medium. The high concentration of compounds constitutes the source of carbon in the culture medium inhibited the biosynthesis of carotenoid pigments by the R. gracilis. Their content in the biomass after cultivation in experimental media with glycerol was more than three times lower (34.6–40.9 μg·g⁻¹ d.m.) to compare with control medium. In these conditions, the yeast synthesized maily torulene (66.9–69.7%) and β-carotene (26.4–29.7%). It was found that the tested yeast strain in the future may become a new source of carotenoids, because of the low volumetric productivity of these compounds (0.9⁻¹.23 mg·dm⁻³), the further studies should be about the effect of the optimization of the culture conditions to increase the efficiency of carotenoids biosynthesis in the media with potato wastewater and glycerol