Oleaginous yeasts- substrate preference and lipid productivity: a view on the performance of microbial lipid producers

Background Oleaginous yeasts are promising microbial platforms for sustainable, bio-based production of biofuels and oleochemical building blocks. Bio-based residues provide sustainable and cost-effective carbon sources for fermentative yeast oil production without land-use change. Considering the regional abundancy of different waste streams, we chose complex biomass residue streams of marine origin; macroalgae hydrolysate, and terrestrial origin; wheat straw hydrolysate in the presence, and absence of corn steep liquor as a complex nitrogen source. We investigated the biomass and lipid yields of an array of well-described oleaginous yeasts; R. glutinis, T. asahii, R. mucilaginosa, R. toruloides, C. oleaginosus growing on these hydrolysates. Furthermore, their sugar utilization, fatty acid profile, and inhibitory effect of the hydrolysates on yeast growth were compared. For correlative reference, we initially performed comparative growth experiments for the strains on individual monomeric sugars separately. Each of these monomeric sugars was a dominant carbon source in the complex biomass hydrolysates evaluated in this study. In addition, we evaluated N-acetylglucosamine, the monomeric building block of chitin, as a low-cost nitrogen and carbon source in yeast fermentation. Results C. oleaginosus provided the highest biomass and lipid yields. In the wheat straw and brown algae hydrolysates, this yeast strain gained 7.5 g/L and 3.8 g/L lipids, respectively. Cultivation in algae hydrolysate resulted in a higher level of unsaturated fatty acids in the lipids accumulated by all yeast strains. R. toruloides and C. oleaginosus were able to effectively co-utilize mannitol, glucose, and xylose. Growth rates on wheat straw hydrolysate were enhanced in presence of corn steep liquor. Conclusions Among the yeast strains investigated in this study, C. oleaginosus proved to be the most versatile strain in terms of substrate utilization, productivity, and tolerance in the complex media. Various fatty acid profiles obtained on each substrate encourage the manipulation of culture conditions to achieve the desired fatty acid composition for each application. This could be accomplished by combining the element of carbon source with other formerly studied factors such as temperature and oxygen. Moreover, corn steep liquor showed promise for enhancement of growth in the oleaginous strains provided that carbon substrate is available.

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.

Improving bio-butanol production from lignocellulosic feedstock by tailoring metabolic perturbations

The objective of this study is to enhance bio-butanol production using lignocellulosic feedstock via supplements of metabolism perturbation. Metabolic perturbations are non-substrate-based chemical additives that can reinforce metabolic flux towards butanol formation, or increase tolerance to microbial inhibitors in the feedstock. Typical metabolic perturbations include CaCO3, ZnSO4, methyl red, and furan derivatives such as furfural and hydroxymethylfurfural (HMF). In this study, we stepwise tailored metabolic perturbations to maximize butanol production from pure sugar and lignocellulosic feedstock. Under optimized conditions of 4 g/L CaCO3, 2 mg/L ZnSO4, butanol production exceeded 10g/L in wheat straw hydrolysate, which was significantly higher than that obtained in the absent of ZnSO4 and CaCO3. As compared to traditional lignocellulosic feedstock post-treatment method, metabolic perturbations method shows advantages in terms of productivity and economics. Improved bio-butanol production is related to the overexpression of NAD(P)H dependent genes.

Improving bio-butanol production from lignocellulosic feedstock by tailoring metabolic perturbations

The objective of this study is to enhance bio-butanol production using lignocellulosic feedstock via supplements of metabolism perturbation. Metabolic perturbations are non-substrate-based chemical additives that can reinforce metabolic flux towards butanol formation, or increase tolerance to microbial inhibitors in the feedstock. Typical metabolic perturbations include CaCO3, ZnSO4, methyl red, and furan derivatives such as furfural and hydroxymethylfurfural (HMF). In this study, we stepwise tailored metabolic perturbations to maximize butanol production from pure sugar and lignocellulosic feedstock. Under optimized conditions of 4 g/L CaCO3, 2 mg/L ZnSO4, butanol production exceeded 10g/L in wheat straw hydrolysate, which was significantly higher than that obtained in the absent of ZnSO4 and CaCO3. As compared to traditional lignocellulosic feedstock post-treatment method, metabolic perturbations method shows advantages in terms of productivity and economics. Improved bio-butanol production is related to the overexpression of NAD(P)H dependent genes.

Ethanol Production from Wheat Straw Hydrolysate by Issatchenkia Orientalis Isolated from Waste Cooking Oil

The interest in using non-conventional yeasts to produce value-added compounds from low cost substrates, such as lignocellulosic materials, has increased in recent years. Setting out to discover novel microbial strains that can be used in biorefineries, an Issatchenkia orientalis strain was isolated from waste cooking oil (WCO) and its capability to produce ethanol from wheat straw hydrolysate (WSHL) was analyzed. As with previously isolated I. orientalis strains, WCO-isolated I. orientalis KJ27-7 is thermotolerant. It grows well at elevated temperatures up to 42 °C. Furthermore, spot drop tests showed that it is tolerant to various chemical fermentation inhibitors that are derived from the pre-treatment of lignocellulosic materials. I. orientalis KJ27-7 is particularly tolerant to acetic acid (up to 75 mM) and tolerates 10 mM formic acid, 5 mM furfural and 10 mM hydroxymethylfurfural. Important for biotechnological cellulosic ethanol production, I. orientalis KJ27-7 grows well on plates containing up to 10% ethanol and media containing up to 90% WSHL. As observed in shake flask fermentations, the specific ethanol productivity correlates with WSHL concentrations. In 90% WSHL media, I. orientalis KJ27-7 produced 10.3 g L−1 ethanol within 24 h. This corresponds to a product yield of 0.50 g g−1 glucose (97% of the theoretical maximum) and a volumetric productivity of 0.43 g L−1 h−1. Therefore, I. orientalis KJ27-7 is an efficient producer of lignocellulosic ethanol from WSHL.

Optimization of steam explosion parameters for improved biotechnological use of wheat straw

Using lignocellulosic raw materials as substrate for biotechnological applications has been a focus of research during the last two decades. They contain sugars, which can be used in industrial fermentation processes, in from of polysaccharides (cellulose, hemicellulose). Wheat straw, one representative of lignocellulosic materials, is sustainably and abundantly available, especially in Europe and North America. However, wheat straw, just like any other lignocellulosic material, needs to be pretreated in one way or the other in order to generate sufficient quantities of monosaccharides. One widely used pretreatment for lignocellulosic material is steam explosion combined with enzymatic hydrolysis. In this study, the effects of steam exploding wheat straw in combination with water are presented. By impregnation with water, saccharide yields from subsequent enzymatic hydrolysis increased from 18.8 to 22.6 g L−1 for glucose and 13.8 to 16.4 g L−1 for xylose, respectively. Moreover, the basic steam explosion parameters residence time and temperature were optimized in ranges from 5 to 20 min and 180–200 °C. This further optimization increased the maximum saccharide yield to 41.2 g L−1 for glucose (200 °C, 15 min) and 18.9 g L−1 for xylose (190 °C, 10 min). Finally, the growth of the intensively investigated biotechnological production host Yarrowia lipolytica on hydrolysates derived from different steam explosion parameters was evaluated. Y. lipolytica grew well in media containing up to 90% wheat straw hydrolysate as sole carbon source, demonstrating the potential as substrate for biotechnological processes.

Metabolic engineering of Aspergillus niger via ribonucleoprotein-based CRISPR–Cas9 system for succinic acid production from renewable biomass

Background Succinic acid has great potential to be a new bio-based building block for deriving a number of value-added chemicals in industry. Bio-based succinic acid production from renewable biomass can provide a feasible approach to partially alleviate the dependence of global manufacturing on petroleum refinery. To improve the economics of biological processes, we attempted to explore possible solutions with a fungal cell platform. In this study, Aspergillus niger, a well-known industrial production organism for bio-based organic acids, was exploited for its potential for succinic acid production. Results With a ribonucleoprotein (RNP)-based CRISPR–Cas9 system, consecutive genetic manipulations were realized in engineering of the citric acid-producing strain A. niger ATCC 1015. Two genes involved in production of two byproducts, gluconic acid and oxalic acid, were disrupted. In addition, an efficient C4-dicarboxylate transporter and a soluble NADH-dependent fumarate reductase were overexpressed. The resulting strain SAP-3 produced 17 g/L succinic acid while there was no succinic acid detected at a measurable level in the wild-type strain using a synthetic substrate. Furthermore, two cultivation parameters, temperature and pH, were investigated for their effects on succinic acid production. The highest amount of succinic acid was obtained at 35 °C after 3 days, and low culture pH had inhibitory effects on succinic acid production. Two types of renewable biomass were explored as substrates for succinic acid production. After 6 days, the SAP-3 strain was capable of producing 23 g/L and 9 g/L succinic acid from sugar beet molasses and wheat straw hydrolysate, respectively. Conclusions In this study, we have successfully applied the RNP-based CRISPR–Cas9 system in genetic engineering of A. niger and significantly improved the succinic acid production in the engineered strain. The studies on cultivation parameters revealed the impacts of pH and temperature on succinic acid production and the future challenges in strain development. The feasibility of using renewable biomass for succinic acid production by A. niger has been demonstrated with molasses and wheat straw hydrolysate.