Production of single cell oil from cane molasses by Rhodotorula kratochvilovae (syn, Rhodosporidium kratochvilovae) SY89 as a biodiesel feedstock

Abstract Background Single cell oil has long been considered an alternative to conventional oil sources. The oil produced can also be used as a feedstock for biodiesel production. Oleaginous yeasts have relatively high growth and lipid production rates, can utilize a wide variety of cheap agro-industrial wastes such as molasses, and can accumulate lipids above 20% of their biomass when they are grown in a bioreactor under conditions of controlled excess carbon and nitrogen limitation. Results In this study, Rhodotorula kratochvilovae (syn, Rhodosporidium kratochvilovae) SY89 was cultivated in a nitrogen-limited medium containing cane molasses as a carbon source. The study aims to provide not only information on the production of single cell oil using R. kratochvilovae SY89 on cane molasses as a biodiesel feedstock, but also to characterize the biodiesel obtained from the resultant lipids. After determination of the sugar content in cane molasses, R. kratochvilovae SY89 was grown on the optimized cane molasses for 168 h. Under the optimized conditions, the yeast accumulated lipids up to 38.25 ± 1.10% on a cellular dry biomass basis. This amount corresponds to a lipid yield of 4.82 ± 0.27 g/L. The fatty acid profiles of the extracted yeast lipids were analyzed using gas chromatography, coupled with flame ionization detector. A significant amount of oleic acid (58.51 ± 0.76%), palmitic acid (15.70 ± 1.27%), linoleic acid (13.29 ± 1.18%) and low amount of other fatty acids were detected in the extracted yeast lipids. The lipids were used to prepare biodiesel and the yield was 85.30%. The properties of this biodiesel were determined and found to be comparable to the specifications established by ASTM D6751 and EN14214 related to biodiesel quality. Conclusions Based on the results obtained, the biodiesel from R. kratochvilovae SY89 oil could be a competitive alternative to conventional diesel fuel

Optimization of cultivation conditions for biotechnological production of lipid by Rhodotorula kratochvilovae (syn, Rhodosporidium kratochvilovae) SY89 for biodiesel preparation

Rhodotorula kratochvilovae (syn, Rhodosporidium kratochvilovae) SY89, an oleaginous yeast, isolated from Ethiopian soil, was grown under nitrogen-limited media. The capacity this with respect to biomass production, lipid yield and lipid content was evaluated. The influence of inoculum size, carbon sources, variations in glucose concentration, nitrogen sources, C/N ratio, pH, temperature, agitation, and aeration rate and incubation period were investigated. Inoculum size of 10% v/v, glucose as a carbon source at 50 g/L glucose, 0.50 g/L yeast extract and 0.31 g/L (NH4)2SO4, C/N ratio of 120, pH 5.5, incubation temperature of 30 °C, 225 rpm, 0.2 as aeration ratio and 144 h of incubation were found to be optimum conditions for lipid production. Then the yeast was grown in a batch bioreactor by combining the different optimized parameters together. Under the optimized conditions, the yeast gave maximum biomass (15.34 ± 1.47 g/L), lipid yield (8.60 ± 0.81 g/L) and lipid content (56.06 ± 1.70%). The dominant fatty acids exhibited in order of their relative abundance (%w/w), were oleic, palmitic, linoleic, stearic, linolenic and palmitoleic acids. The concentration of saturated and monounsaturated fatty acids adds up 78.63 ± 2.19%. This suggests that this strain could be used as a good feedstock for biodiesel production

The Hormetic Effect Observed for Benzalkonium Chloride and Didecyldimethylammonium Chloride in <i>Serratia</i> sp. HRI

Hormesis, or the hormetic effect, is a dose- or concentration-dependent response characterised by growth stimulation at low concentrations and inhibition at high concentrations. The impact of sub-lethal levels of disinfectants on the growth of Serratia species is critical to understanding the increasing number of outbreaks caused by this pathogen in healthcare settings. Serratia sp. HRI and Serratia marcescens ATCC 13880 were cultivated in sub-lethal levels of benzalkonium chloride (BAC), Didecyldimethylammonium chloride (DDAC), and VirukillTM. The maximum specific growth rates, doubling times, and cell counts were compared. The results revealed significant increases in maximum specific growth rates and shorter doubling times for Serratia sp. HRI when cultivated in sub-lethal levels of BAC and DDAC. The significant stimulatory effect of sub-lethal levels of these disinfectants for Serratia sp. HRI represents the first time hormesis has been observed in a Gram-negative bacterium for any disinfectant. Furthermore, this study is the first to observe the hormetic effect after treatment with DDAC and the second study to date analysing the impact of sub-lethal levels of disinfectants on the growth of bacterial species

Differential RNA-seq, Multi-Network Analysis and Metabolic Regulation Analysis of Kluyveromyces marxianus Reveals a Compartmentalised Response to Xylose.

We investigated the transcriptomic response of a new strain of the yeast Kluyveromyces marxianus, in glucose and xylose media using RNA-seq. The data were explored in a number of innovative ways using a variety of networks types, pathway maps, enrichment statistics, reporter metabolites and a flux simulation model, revealing different aspects of the genome-scale response in an integrative systems biology manner. The importance of the subcellular localisation in the transcriptomic response is emphasised here, revealing new insights. As was previously reported by others using a rich medium, we show that peroxisomal fatty acid catabolism was dramatically up-regulated in a defined xylose mineral medium without fatty acids, along with mechanisms to activate fatty acids and transfer products of β-oxidation to the mitochondria. Notably, we observed a strong up-regulation of the 2-methylcitrate pathway, supporting capacity for odd-chain fatty acid catabolism. Next we asked which pathways would respond to the additional requirement for NADPH for xylose utilisation, and rationalised the unexpected results using simulations with Flux Balance Analysis. On a fundamental level, we investigated the contribution of the hierarchical and metabolic regulation levels to the regulation of metabolic fluxes. Metabolic regulation analysis suggested that genetic level regulation plays a major role in regulating metabolic fluxes in adaptation to xylose, even for the high capacity reactions, which is unexpected. In addition, isozyme switching may play an important role in re-routing of metabolic fluxes in subcellular compartments in K. marxianus

2-Methylcitrate pathway.

<p>The suggested route of three-carbon units is indicated in magenta.</p

Histogram of RNA-seq log₂(fold change) values.

<p>Statistical procedures were performed in CuffDiff.</p

Total transcript levels in central metabolic pathways with glucose as the carbon source.

<p>Transcript levels for all genes catalysing a reaction were summed. Note the logarithmic scale. Dark grey indicates genes present and constitutively expressed. Light grey indicates genes not found in annotation or combined reactions. For reaction names, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156242#pone.0156242.s008" target="_blank">S1 Pathway</a>.</p

Uncompartmentalised response to xylose in central carbon metabolism as a classification scheme.

<p>Blue reactions represent those for which more than one enzyme gene has been assigned and for which some were up-regulated and some down-regulated, referred to as isozyme switching. For reaction names, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156242#pone.0156242.s008" target="_blank">S1 Pathway</a>.</p

Uncompartmentalised response to xylose in central carbon metabolism in a log₂(fold change) scheme.

<p>A log₂(fold change) is defined as the log₂ ratio of transcripts on xylose divided by that on glucose, as reported by CuffDiff. Reactions vETC, vEthylAcetate and vGrowth were manually added to the model. In the case of more than one enzyme that could perform the same function, the largest fold change in expression was used for the colour rendering. For reaction names, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156242#pone.0156242.s008" target="_blank">S1 Pathway</a>.</p

Enzyme-metabolite interaction network around redox cofactors.

<p>Left: NADH; Right: NADP⁺. For reporter metabolites (circles), size indicates the enrichment score of a gene set and colour indicates the up/down direction of regulation. Brightness indicates uni-directionality of regulation. For enzymes (stars), colour indicates the up/down direction of regulation based on the log₂(fold change) scheme. (For full information on gene names, the interactive file <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156242#pone.0156242.s005" target="_blank">S4 Network</a> or the corresponding annotations in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156242#pone.0156242.s012" target="_blank">S1 Table</a>, using the “Gene names (primary)” column, may be consulted.)</p