Detektion och kvantifiering av mögel på spannmål med realtids PCR

Tillväxt av mögel och jäst är ett problem vid lagring av spannmål ensilage och andra fodermedel. Traditionella metoder för identifiering och kvantifiering av sådana mikrosvampar är tidskrävande och mödosamma. Det finns därför ett behov för att utveckla nya, snabba och pålitliga förfaranden för identifiering och kvantifiering. Vi har utvecklat en molekylär metod som kan används för kvantifiering av mögel och jäst som växer på spannmål eller andra fodermedel. Förfarandet är baserat på direkt isolering av mikroorganismernas DNA från spannmål och kvantifiering av organismspecifika gener med realtids-PCR. Metoden utprovades i ett projekt för odling av mycelväxade svamp (Rhizopus oligosporus) och jäst (Saccharomyces cerevisiae) på korn. En enkel DNA-extraktion var effektiv för mögel, jäst och även mjölksyrabakterier. Koncentrationen av organismspecifika gener (determinerade i realtids PCR) korrelerade med ergosterolhalten (som indikerar svamptillväxt) i R. oligosporus ren kultur och med antal kolonibildande enheter av jäst och mjölksyrabakterier. Detektionen var mycket specifik för de testade organism, och ingen korsdetektion upptäcktes. Förfarandet är snabbt och pålitligt med möjlighetet till analys av 50 prover under två dagar. Metoden kan antagligen användas för detektion och kvantifiering av även andra mögelarter på andra sorts spannmål. Man kan även identifiera specifika gener, t.ex. gener som är involverade i mykotoxinproduktionen

From straw to salmon: a technical design and energy balance for production of yeast oil for fish feed from wheat straw

BackgroundAquaculture is a major user of plant-derived feed ingredients, such as vegetable oil. Production of vegetable oil and protein is generally more energy-intensive than production of the marine ingredients they replace, so increasing inclusion of vegetable ingredients increases the energy demand of the feed. Microbial oils, such as yeast oil made by fermentation of lignocellulosic hydrolysate, have been proposed as a complement to plant oils, but energy assessments of microbial oil production are needed. This study presents a mass and energy balance for a biorefinery producing yeast oil through conversion of wheat straw hydrolysate, with co-production of biomethane and power.ResultsThe results showed that 1 tonne of yeast oil (37 GJ) would require 9.2 tonnes of straw, 14.7 GJ in fossil primary energy demand, 14.6 GJ of process electricity and 13.3 GJ of process heat, while 21.5 GJ of biomethane (430 kg) and 6 GJ of excess power would be generated simultaneously. By applying economic allocation, the fossil primary energy demand was estimated to 11.9 GJ per tonne oil.ConclusionsFossil primary energy demand for yeast oil in the four scenarios studied was estimated to be 10-38% lower than for the commonly used rapeseed oil and process energy demand could be met by parallel combustion of lignin residues. Therefore, feed oil can be produced from existing non-food biomass without causing agricultural expansion

Greenhouse gas performance of biodiesel production from straw: Soil carbon changes and time-dependent climate impact

Background: Use of bio‑based diesel is increasing in Europe. It is currently produced from oilseed crops, but can also be generated from lignocellulosic biomass such as straw. However, removing straw affects soil organic carbon (SOC), with potential consequences for the climate impact of the biofuel. This study assessed the climate impacts and energy balance of biodiesel production from straw using oleaginous yeast, with subsequent biogas production from the residues, with particular emphasis on SOC changes over time. It also explored the impact of four different scenarios for returning the lignin fraction of the biomass to soil to mitigate SOC changes. Climate impact was assessed using two methods, global warming potential (GWP) and a time‑dependent temperature model (∆Ts) that describes changes in mean global surface temperature as a function of time or absolute temperature change potential (AGTP). Results: Straw‑derived biodiesel reduced GWP by 33–80% compared with fossil fuels and primary fossil energy use for biodiesel production was 0.33–0.80 MJprim/MJ, depending on the scenario studied. Simulations using the time‑dependent temperature model showed that a scenario where all straw fractions were converted to energy carriers and no lignin was returned to soil resulted in the highest avoided climate impact. The SOC changes due to straw removal had a large impact on the results, both when using GWP and the time‑dependent temperature model. Conclusions: In a climate perspective, it is preferable to combust straw lignin to produce electricity rather than returning it to the soil if the excess electricity replaces natural gas electricity, according to results from both GWP and time‑dependent temperature modelling. Using different methods to assess climate impact did not change the ranking between the scenarios, but the time‑dependent temperature model provided information about system behaviour over time that can be important for evaluation of biofuel systems, particularly in relation to climate target deadlines

Near Chromosome-Level Genome Assembly and Annotation of Rhodotorula babjevae Strains Reveals High Intraspecific Divergence

The genus Rhodotorula includes basidiomycetous oleaginous yeast species. Rhodotorula babjevae can produce compounds of biotechnological interest such as lipids, carotenoids, and biosurfactants from low value substrates such as lignocellulose hydrolysate. High-quality genome assemblies are needed to develop genetic tools and to understand fungal evolution and genetics. Here, we combined short- and long-read sequencing to resolve the genomes of two R. babjevae strains, CBS 7808 (type strain) and DBVPG 8058, at chromosomal level. Both genomes are 21 Mbp in size and have a GC content of 68.2%. Allele frequency analysis indicates that both strains are tetraploid. The genomes consist of a maximum of 21 chromosomes with a size of 0.4 to 2.4 Mbp. In both assemblies, the mitochondrial genome was recovered in a single contig, that shared 97% pairwise identity. Pairwise identity between most chromosomes ranges from 82 to 87%. We also found indications for strain-specific extrachromosomal endogenous DNA. A total of 7591 and 7481 protein-coding genes were annotated in CBS 7808 and DBVPG 8058, respectively. CBS 7808 accumulated a higher number of tandem duplications than DBVPG 8058. We identified large translocation events between putative chromosomes. Genome divergence values between the two strains indicate that they may belong to different species.Peer Reviewe

Oleaginous yeasts respond differently to carbon sources present in lignocellulose hydrolysate

Background Microbial oils, generated from lignocellulosic material, have great potential as renewable and sustainable alternatives to fossil-based fuels and chemicals. By unravelling the diversity of lipid accumulation physiology in different oleaginous yeasts grown on the various carbon sources present in lignocellulose hydrolysate (LH), new targets for optimisation of lipid accumulation can be identified. Monitoring lipid formation over time is essential for understanding lipid accumulation physiology. This study investigated lipid accumulation in a variety of oleaginous ascomycetous and basidiomycetous strains grown in glucose and xylose and followed lipid formation kinetics of selected strains in wheat straw hydrolysate (WSH). Results Twenty-nine oleaginous yeast strains were tested for their ability to utilise glucose and xylose, the main sugars present in WSH. Evaluation of sugar consumption and lipid accumulation revealed marked differences in xylose utilisation capacity between the yeast strains, even between those belonging to the same species. Five different promising strains, belonging to the species Lipomyces starkeyi, Rhodotorula glutinis, Rhodotorula babjevae and Rhodotorula toruloides, were grown on undiluted wheat straw hydrolysate and lipid accumulation was followed over time, using Fourier transform-infrared (FTIR) spectroscopy. All five strains were able to grow on undiluted WSH and to accumulate lipids, but to different extents and with different productivities. R. babjevae DVBPG 8058 was the best-performing strain, accumulating 64.8% of cell dry weight (CDW) as lipids. It reached a culture density of 28 g/L CDW in batch cultivation, resulting in a lipid content of 18.1 g/L and yield of 0.24 g lipids per g carbon source. This strain formed lipids from the major carbon sources in hydrolysate, glucose, acetate and xylose. R. glutinis CBS 2367 also consumed these carbon sources, but when assimilating xylose it consumed intracellular lipids simultaneously. Rhodotorula strains contained a higher proportion of polyunsaturated fatty acids than the two tested Lipomyces starkeyi strains. Conclusions There is considerable metabolic diversity among oleaginous yeasts, even between closely related species and strains, especially when converting xylose to biomass and lipids. Monitoring the kinetics of lipid accumulation and identifying the molecular basis of this diversity are keys to selecting suitable strains for high lipid production from lignocellulose

Montana Kaimin, January 30, 2008

Student newspaper of the University of Montana, Missoula.https://scholarworks.umt.edu/studentnewspaper/6138/thumbnail.jp

Enhanced glycerol assimilation and lipid production in Rhodotorula toruloides CBS14 upon addition of hemicellulose primarily correlates with early transcription of energy-metabolism-related genes

BackgroundLipid formation from glycerol was previously found to be activated in Rhodotorula toruloides when the yeast was cultivated in a mixture of crude glycerol (CG) and hemicellulose hydrolysate (CGHH) compared to CG as the only carbon source. RNA samples from R. toruloides CBS14 cell cultures grown on either CG or CGHH were collected at different timepoints of cultivation, and a differential gene expression analysis was performed between cells grown at a similar physiological situation.ResultsWe observed enhanced transcription of genes involved in oxidative phosphorylation and enzymes localized in mitochondria in CGHH compared to CG. Genes involved in protein turnover, including those encoding ribosomal proteins, translation elongation factors, and genes involved in building the proteasome also showed an enhanced transcription in CGHH compared to CG. At 10 h cultivation, another group of activated genes in CGHH was involved in beta-oxidation, handling oxidative stress and degradation of xylose and aromatic compounds. Potential bypasses of the standard GUT1 and GUT2-glycerol assimilation pathway were also expressed and upregulated in CGHH 10 h. When the additional carbon sources from HH were completely consumed, at CGHH 36 h, their transcription decreased and NAD(+)-dependent glycerol-3-phosphate dehydrogenase was upregulated compared to CG 60 h, generating NADH instead of NADPH with glycerol catabolism. TPI1 was upregulated in CGHH compared to cells grown on CG in all physiological situations, potentially channeling the DHAP formed through glycerol catabolism into glycolysis. The highest number of upregulated genes encoding glycolytic enzymes was found after 36 h in CGHH, when all additional carbon sources were already consumed.ConclusionsWe suspect that the physiological reason for the accelerated glycerol assimilation and faster lipid production, was primarily the activation of enzymes that provide energy

Screening of intact yeasts and cell extracts to reduce Scrapie prions during biotransformation of food waste

Yeasts can be used to convert organic food wastes to protein-rich animal feed in order to recapture nutrients. However, the reuse of animal-derived waste poses a risk for the transmission of infectious prions that can cause neurodegeneration and fatality in humans and animals. The aim of this study was to investigate the ability of yeasts to reduce prion activity during the biotransformation of waste substrates—thereby becoming a biosafety hurdle in such a circular food system. During pre-screening, 30 yeast isolates were spiked with Classical Scrapie prions and incubated for 72 h in casein substrate, as a waste substitute. Based on reduced Scrapie seeding activity, waste biotransformation and protease activities, intact cells and cell extracts of 10 yeasts were further tested. Prion analysis showed that five yeast species reduced Scrapie seeding activity by approximately 1 log10 or 90%. Cryptococcus laurentii showed the most potential to reduce prion activity since both intact and extracted cells reduced Scrapie by 1 log10 and achieved the highest protease activity. These results show that select forms of yeast can act as a prion hurdle during the biotransformation of waste. However, the limited ability of yeasts to reduce prion activity warrants caution as a sole barrier to transmission as higher log reductions are needed before using waste-cultured yeast in circular food systems

Identification, Quantification and Kinetic Study of Carotenoids and Lipids in Rhodotorula toruloides CBS 14 Cultivated on Wheat Straw Hydrolysate

Production of carotenoids and lipids by Rhodotorula toruloides CBS 14 cultivated on wheat straw hydrolysate was investigated. An ultra-high-performance liquid chromatography (UHPLC) method for carotenoid quantification was developed and validated. Saponification effects on individual carotenoid quantification were identified, and lipid and carotenoid kinetics during cultivation were determined. The carotenoids beta-carotene, gamma-carotene, torularhodin, and torulene were identified; beta-carotene was the major carotenoid, reaching a maximum of 1.48 mg/100 g dry weight. Recoveries of the carotenoids were between 66% and 76%, except torulene and torularhodin, which had lower recoveries due to saponification effects. Total carotenoid content in saponified and unsaponified yeast extract, respectively, determined by UHPLC or photometer, respectively, was 1.99 mg/100 g and 4.02 mg beta-EQ/100 g dry weight. Growth kinetics showed a positive correlation between carotenoid content and lipid accumulation. beta-carotene was the major carotenoid at all time points. At the end of the cultivation, triacylglycerols (TAGs) were the major lipid class, with 58.1% +/- 3.32% of total lipids. There was also a high proportion of free fatty acids, reaching from 20.5% to 41.8% of total lipids. Oleic acid (C18:1) was the major fatty acid. The lipid yield at the end of the cultivation was 0.13 g/g of sugar consumed