Oleaginous yeasts for biochemicals, biofuels and food from lignocellulose-hydrolysate and crude glycerol.

Microbial lipids produced from lignocellulose and crude glycerol (CG) can serve as sustainable alternatives to vegetable oils, whose production is, in many cases, accompanied by monocultures, land use changes or rain forest clearings. Our projects aim to understand the physiology of microbial lipid production by oleaginous yeasts, optimise the production and establish novel applications of microbial lipid compounds. We have established methods for fermentation and intracellular lipid quantification. Following the kinetics of lipid accumulation in different strains, we found high variability in lipid formation even between very closely related oleaginous yeast strains on both, wheat straw hydrolysate and CG. For example, on complete wheat straw hydrolysate, we saw that one Rhodotorula glutinis strain, when starting assimilating D-xylosealso assimilated the accumulated lipids, while a Rhodotorula babjevae strain could accumulate lipids on D-xylose. Two strains (Rhodotorula toruloides CBS 14 and R. glutinis CBS 3044) were found to be the best out of 27 tested to accumulate lipids on CG. Interestingly, the presence of hemicellulose hydrolysate stimulated glycerol assimilation in both strains. Apart from microbial oil, R. toruloides also produces carotenoids. The first attempts of extraction using the classical acetone-based method showed that ß-carotene is the major carotenoid. However, there are indications that there are also substantial amounts of torulene and torularhodin, which have a very high potential as antioxidants.

Chromosome-level genome assembly and transcriptome-based annotation of the oleaginous yeast Rhodotorula toruloides CBS 14.

Rhodotorula toruloides is an oleaginous yeast with high biotechnological potential. In order to understand the molecular physiology of lipid synthesis in R. toruloides and to advance metabolic engineering, a high-resolution genome is required. We constructed a genome draft of R. toruloides CBS 14, using a hybrid assembly approach, consisting of short and long reads generated by Illumina and Nanopore sequencing, respectively. The genome draft consists of 23 contigs and 3 scaffolds, with a N50 length of 1,529,952 bp, thus largely representing chromosomal organization. The total size of the genome is 20,534,857 bp and the overall GC content is 61.83%. Transcriptomic data from different growth conditions was used to aid species-specific gene annotation. We annotated 9464 genes and identified 11,691 transcripts. Furthermore, we demonstrated the presence of a potential plasmid, an extrachromosomal circular structure of about 11 kb with a copy number about three times as high as the other chromosomes.

Insertional tagging of the Scheffersomyces stipitis gene HEM25 involved in regulation of glucose and xylose alcoholic fermentation.

Amid known microbial bioethanol producers, the yeast Scheffersomyces (Pichia) stipitis is particularly promising in terms of alcoholic fermentation of both glucose and xylose, the main constituents of lignocellulosic biomass hydrolysates. However, the ethanol yield and productivity, especially from xylose, are still insufficient to meet the requirements of a feasible industrial technology; therefore, the construction of more efficient S. stipitis ethanol producers is of great significance. The aim of this study was to isolate the insertional mutants of S. stipitis with altered ethanol production from glucose and xylose and to identify the disrupted gene(s). Mutants obtained by random insertional mutagenesis were screened for their growth abilities on solid media with different sugars and for resistance to 3-bromopyruvate. Of more than 1,300 screened mutants, 17 were identified to have significantly changed ethanol yields during the fermentation. In one of the best fermenting strains (strain 4.6), insertion was found to occur within the ORF of a homolog to the Saccharomyces cerevisiae gene HEM25 (YDL119C), encoding a mitochondrial glycine transporter required for heme synthesis. The role of HEM25 in heme accumulation, respiration, and alcoholic fermentation in the yeast S. stipitis was studied using strain 4.6, the complementation strain Comp-a derivative from the 4.6 strain with expression of the WT HEM25 allele and the deletion strain hem25Δ. As hem25Δ produced lower amounts of ethanol than strain 4.6, we assume that the phenotype of strain 4.6 may be caused not only by HEM25 disruption but additionally by some point mutation.

Yeasts of the Blastobotrys genus are promising platform for lipid-based fuels and oleochemicals production.

Strains of the yeast genus Blastobotrys (subphylum Saccharomycotina) represent a valuable biotechnological resource for basic biochemistry research, single-cell protein, and heterologous protein production processes. Species of this genus are dimorphic, non-pathogenic, thermotolerant, and can assimilate a variety of hydrophilic and hydrophobic substrates. These can constitute a single-cell oil platform in an emerging bio-based economy as oleaginous traits have been discovered recently. However, the regulatory network of lipogenesis in these yeasts is poorly understood. To keep pace with the growing market demands for lipid-derived products, it is critical to understand the lipid biosynthesis in these unconventional yeasts to pinpoint what governs the preferential channelling of carbon flux into lipids instead of the competing pathways. This review summarizes information relevant to the regulation of lipid metabolic pathways and prospects of metabolic engineering in Blastobotrys yeasts for their application in food, feed, and beyond, particularly for fatty acid-based fuels and oleochemicals. KEY POINTS: • The production of biolipids by heterotrophic yeasts is reviewed. • Summary of information concerning lipid metabolism regulation is highlighted. • Special focus on the importance of diacylglycerol acyltransferases encoding genes in improving lipid production is made.

Production and characterization of yeasts grown on media composed of spruce-derived sugars and protein hydrolysates from chicken by-products.

BACKGROUND: A possible future shortage of feed protein will force mankind to explore alternative protein sources that can replace conventional soymeal or fishmeal. Several large industrial organic side-streams could potentially be upgraded to feed protein using a fermentation process to generate single cell protein. Yeast is the most widely accepted microorganism for production of single cell protein, because of its superior nutritional quality and acceptability among consumers. Here, we have assessed the growth of four different yeasts, Cyberlindnera jadinii, Wickerhamomyces anomalus, Blastobotrys adeninivorans and Thermosacc® Dry (Saccharomyces cerevisiae), on media composed of enzymatically saccharified sulfite-pulped spruce wood and hydrolysates of by-products from chicken, and we have characterized the resulting yeast biomass. RESULTS: Generally, the yeast grew very well on the spruce- and chicken-based medium, with typical yields amounting to 0.4-0.5 g of cell dry weight and 0.2-0.3 g of protein per g of sugar. B. adeninivorans stood out as the most versatile yeast in terms of nutrient consumption and in this case yields were as high as 0.9 g cells and 0.5 g protein per g of sugar. The next best performing yeast in terms of yield was W. anomalus with up to 0.6 g cells and 0.3 g protein per g sugar. Comparative compositional analyses of the yeasts revealed favorable amino acid profiles that were similar to the profiles of soymeal, and even more so, fish meal, especially for essential amino acids. CONCLUSIONS: The efficient conversion of industrial biomass streams to yeast biomass demonstrated in this study opens new avenues towards better valorization of these streams and development of sustainable feed ingredients. Furthermore, we conclude that production of W. anomalus or B. adeninivorans on this promising renewable medium may be potentially more efficient than production of the well-known feed ingredient C. jadinii. Further research should focus on medium optimization, development of semi-continuous and continues fermentation protocols and exploration of downstream processing methods that are beneficial for the nutritional values of the yeast for animal feed.

Spruce sugars and poultry hydrolysate as growth medium in repeated fed-batch fermentation processes for production of yeast biomass.

The production of microbial protein in the form of yeast grown on lignocellulosic sugars and nitrogen-rich industrial residues is an attractive approach for reducing dependency on animal and plant protein. Growth media composed of enzymatically saccharified sulfite-pulped spruce wood, enzymatic hydrolysates of poultry by-products and urea were used for the production of single-cell protein. Strains of three different yeast species, Cyberlindnera jadinii, Wickerhamomyces anomalus and Blastobotrys adeninivorans, were cultivated aerobically using repeated fed-batch fermentation up to 25 L scale. Wickerhamomyces anomalus was the most efficient yeast with yields of 0.6 g of cell dry weight and 0.3 g of protein per gram of glucose, with cell and protein productivities of 3.92 g/L/h and 1.87 g/L/h, respectively. Using the conditions developed here for producing W. anomalus, it would take 25 industrial (200 m3) continuously operated fermenters to replace 10% of the fish feed protein used in Norway.

Biofuel production from straw hydrolysates: current achievements and perspectives.

Straw is an agricultural residue of the production of e.g. cereals, rapeseed or sunflowers. It includes dried stalks, leaves, and empty ears and corncobs, which are separated from the grains during harvest. Straw is a promising lignocellulosic feedstock with a beneficial greenhouse gas balance for the production of biofuels and chemicals. Like all lignocellulosic materials, straw is recalcitrant and requires thermochemical and enzymatic pretreatment to enable access to the three major biopolymers of straw-the polysaccharides cellulose and hemicellulose and the polyaromatic compound lignin. Straw is used for commercial ethanol and biogas production. Considerable research has also been conducted to produce biobutanol, biodiesel and biochemicals from this raw material, but more research is required to establish them on a commercial scale. The major hindrance for launching industrial biofuel and chemicals' production from straw is the high cost necessitated by pretreatment of the material. Improvements of microbial strains, production and extraction technologies, as well as co-production of high-value compounds represent ways of establishing straw as feedstock for the production of biofuels, chemicals and food.

Yeasts and bacteria associated with kocho, an Ethiopian fermented food produced from enset (Ensete ventricosum).

Enset (Ensete ventricosum) is the basis of the staple food consumed by about 20% of the Ethiopian population. Kocho is one of the food products generated from enset by spontaneous fermentation of decorticated and pulverized pseudostem and corm sections. We isolated culturable microbes associated with kocho from different stages of fermentation. Twelve yeast species, six lactic acid bacteria (LABs) species and eleven species of aerobic bacteria were identified by sequencing ITS/D1D2 regions of 26S rDNA of yeasts and 16S rDNA of bacteria, respectively. More yeast species were identified in fresh (fermented for 2-5 days) kocho, compared to long-term (7-12 months) fermented kocho, while we observed an opposite trend for LABs. In fresh kocho, the most frequently isolated yeast species were Pichia exigua, Galactomyces geotrichum, and Pichia fermentans. From mid-term (3-4 months) kocho most frequently Candida cabralensis, G. geotrichum, and Candida ethanolica were isolated. In the long-term fermentations, the most frequently isolated yeast was Saturnispora silva. Lactobacillus plantarum was the most frequently isolated LAB in both fresh and mid-term kocho. In long-term fermented kocho, Acetobacter pasteurianus and L. plantarum were most frequently isolated. L. plantarum was consistently isolated from all the three stages of fermentation. Aerobic bacteria in fresh kocho were mostly gram-negative, with Raoultella planticola and Pantoea agglomerans being the most frequently isolated species. In long-term fermented kocho, mainly gram-positive, spore-forming bacteria of the genus Bacillus were found, among them also species of the Bacillus cereus group, Bacillus anthracis and Bacillus thurigiensis.

Bioethanol and lipid production from the enzymatic hydrolysate of wheat straw after furfural extraction.

This study investigates biofuel production from wheat straw hydrolysate, from which furfural was extracted using a patented method developed at the Latvian State Institute of Wood Chemistry. The solid remainder after furfural extraction, corresponding to 67.6% of the wheat straw dry matter, contained 69.9% cellulose of which 4% was decomposed during the furfural extraction and 26.3% lignin. Enzymatic hydrolysis released 44% of the glucose monomers in the cellulose. The resulting hydrolysate contained mainly glucose and very little amount of acetic acid. Xylose was not detectable. Consequently, the undiluted hydrolysate did not inhibit growth of yeast strains belonging to Saccharomyces cerevisiae, Lipomyces starkeyi, and Rhodotorula babjevae. In the fermentations, average final ethanol concentrations of 23.85 g/l were obtained, corresponding to a yield of 0.53 g ethanol per g released glucose. L. starkeyi generated lipids with a rate of 0.08 g/h and a yield of 0.09 g per g consumed glucose. R. babjevae produced lipids with a rate of 0.18 g/h and a yield of 0.17 per g consumed glucose. In both yeasts, desaturation increased during cultivation. Remarkably, the R. babjevae strain used in this study produced considerable amounts of heptadecenoic, α,- and γ-linolenic acid.

Lipid production from hemicellulose with Lipomyces starkeyi in a pH regulated fed-batch cultivation.

This study investigated lipid production from the hemicellulosic fraction of birch wood by the oleaginous yeast Lipomyces starkeyi. Birch wood chips were thermochemically pretreated by hot water extraction, and the liquid phase, containing 45.1 g/l xylose as the major sugar, 13.1 g/l acetic acid and 4.7 g/l furfural, was used for cultivations of L. starkeyi CBS1807. The hydrolysate strongly inhibited yeast growth; the strain could only grow in medium containing 30% hydrolysate at pH 6. At pH 5, growth stopped already upon the addition of about 10% hydrolysate. In fed-batch cultures fed with hydrolysate or a model xylose-acetic acid mixture, co-consumption of xylose and acetic acid was observed, which resulted in a pH increase. This phenomenon was utilized to establish a pH-stat fed-batch cultivation in which, after an initial feeding, hydrolysate or model mixture was connected to the pH-regulation system of the bioreactor. Under these conditions we obtained growth and lipid production in cultures grown on either xylose or glucose during the batch phase. In cultivations fed with model mixture, a maximum lipid content of 60.5% of the cell dry weight (CDW) was obtained; however, not all xylose was consumed. When feeding hydrolysate, growth was promoted and carbon sources were completely consumed, resulting in higher CDW with maximum lipid content of 51.3%. In both cultures the lipid concentration was 8 g/l and a lipid yield of 0.1 g/g carbon source was obtained. Lipid composition was similar in all cultivations, with C18:1 and C16:0 being the most abundant fatty acids. Copyright © 2016 John Wiley & Sons, Ltd.

Dekkera bruxellensis--spoilage yeast with biotechnological potential, and a model for yeast evolution, physiology and competitiveness.

Dekkera bruxellensis is a non-conventional yeast normally considered a spoilage organism in wine (off-flavours) and in the bioethanol industry. But it also has potential as production yeast. The species diverged from Saccharomyces cerevisiae 200 mya, before the whole genome duplication. However, it displays similar characteristics such as being Crabtree- and petite positive, and the ability to grow anaerobically. Partial increases in ploidy and promoter rewiring may have enabled evolution of the fermentative lifestyle in D. bruxellensis. On the other hand, it has genes typical for respiratory yeasts, such as for complex I or the alternative oxidase AOX1. Dekkera bruxellensis grows more slowly than S. cerevisiae, but produces similar or greater amounts of ethanol, and very low amounts of glycerol. Glycerol production represents a loss of energy but also functions as a redox sink for NADH formed during synthesis of amino acids and other compounds. Accordingly, anaerobic growth required addition of certain amino acids. In spite of its slow growth, D. bruxellensis outcompeted S. cerevisiae in glucose-limited cultures, indicating a more efficient energy metabolism and/or higher affinity for glucose. This review tries to summarize the latest discoveries about evolution, physiology and metabolism, and biotechnological potential of D. bruxellensis.

Interaction of Lactobacillus vini with the ethanol-producing yeasts Dekkera bruxellensis and Saccharomyces cerevisiae.

Lactobacillus vini was recently described as a contaminant in industrial ethanol fermentations and its co-occurrence with Dekkera bruxellensis was noted. We investigated the growth characteristics of L. vini in cocultivation together with either Saccharomyces cerevisiae or D. bruxellensis. Lower cell numbers of both the yeasts and L. vini as well as a decrease in ethanol and lactate formation in mixed batch cultures compared with pure cultures were noted. L. vini formed cell aggregates (flocs) in all cultivation media with different shapes in Man-Rogosa-Sharpe and yeast extract-peptone-dextrose media. Flocs' size and proportion of cells bound to flocs increased with increasing ethanol concentration. In coculture, formation of lactic acid bacteria-yeast cell aggregates consisting of a bacterial core with an outer layer of yeast cells was observed. L. vini-D. bruxellensis flocs had a bigger surface, due to cells protruding from the pseudomycelium. The involvement of mannose residues in the flocculation between L. vini and yeasts was tested. The presence of mannose induced deflocculation in a concentration-dependent manner. Less mannose was required for the deflocculation of D. bruxellensis as compared with S. cerevisiae.

Strain- and temperature-dependent changes of fatty acid composition in Wickerhamomyces anomalus and Blastobotrys adeninivorans.

The fatty acid (FA) profiles of two strains of the yeasts Wickerhamomyces anomalus and Blastobotrys (Arxula) adeninivorans at cultivation temperatures from 15 to 30 °C were characterized. Besides the common even-numbered C16 and C18 FAs, substantial proportions of the uneven-numbered C17:1 were found in both species. C18:3(n-3) (alpha linolenic acid) made up to 3% of the total FAs in all strains. Considerable strain differences occurred, with regard to both the presence of single FAs and parameters like the double binding index (DBI) and C16:C18 ratio. W. anomalus J121 formed C18:1(n-5) (up to 10.9% of the total FAs) but no C18:1(n-7), whereas in W. anomalus VKM160, no C18:1(n-5) was found but up to 14.6% C18:1(n-7). Similarly, B. adeninivorans CBS 8244 formed exclusively C18:1(n-7) (maximum 9%) and CBS 7377 C18:1(n-5) (maximum 12.6%). W. anomalus J121 had the lowest DBI (0.72) at 15 °C and the highest (0.92) at 20 °C, at which point the values decreased with increasing temperatures. In W. anomalus VKM160 and both B. adeninivorans strains, DBI was highest at 15 °C and decreased with increasing temperature. In J121, the C16:C18 ratio was highest at 15 °C, decreasing at higher temperatures, whereas in the other strains, the opposite trend was observed.

Temperature-dependent changes in the microbial storage flora of birch and spruce sawdust.

Sawdust can be used to make pellets (biofuel) and particle boards and as a potential lignocellulose feedstock in bioethanol production. Microbial activity can affect sawdust quality; hence, we monitored the microbial population in birch- and spruce sawdust after 3 months' storage at various temperatures. Species composition was similar on both materials but was strongly influenced by temperature. Bacteria were present on all materials at all conditions: on birch, 2.8 × 10(8) , 1.1 × 10(8) , and 8.8 × 10(6) , and on spruce, 4.1 × 10(8) , 5.6 × 10(7) , and 1.5 × 10(8)  CFU/g DM, at 2, 20, and 37 °C, respectively. Dominant bacteria at 2, 20, and 37 °C were Pseudomonas spp. (some Enterobacteriaceae spp. present), Luteibacter rhizovicinus, and Fulvimonas sp., respectively. Pseudomonas spp. were absent at ≥20 °C. Among microfungi, yeasts dominated at 2 °C but were absent at 37 °C, whereas molds dominated at 20 and 37 °C. Common yeasts included Cystofilobasidium capitatum, Cystofilobasidium infirmominiatum, Candida saitoana, Candida oregonensis, and Candida railenensis. Ophiostoma quercus was a common mold at 2 and 20 °C, whereas the human pathogens Aspergillus fumigatus and Paecilomyces variotii dominated at 37 °C. Attempts to influence the microflora by addition of the biocontrol yeasts, Wickerhamomyces anomalus and Scheffersomyces stipitis, were unsuccessful, as their growth in sawdust was poor to absent.

Adaptation of Dekkera bruxellensis to lignocellulose-based substrate.

Adaptation of Dekkera bruxellensis to lignocellulose hydrolysate was investigated. Cells of D. bruxellensis were grown for 72 and 192 H in batch and continuous culture, respectively (adapted cells). Cultivations in semisynthetic medium were run as controls (nonadapted cells). To test the adaptation, cells from these cultures were reinoculated in the lignocellulose medium, and growth and ethanol production characteristics were monitored. Cells adapted to lignocellulose hydrolysate had a shorter lag phase, grew faster, and produced a higher ethanol concentration as compared with nonadapted cells. A stability test showed that after cultivation in rich medium, cells partially lost the adapted phenotype but still showed faster growth and higher ethanol production as compared with nonadapted cells. Because alcohol dehydrogenase genes have been described to be involved in the adaptation to furfural in Saccharomyces cerevisiae, an analogous mechanism of adaptation to lignocelluloses hydrolysate of D. bruxellensis was hypothesized. However, gene expression analysis showed that genes homologous to S. cerevisiae ADH1 were not involved in the adaptation to lignocelluloses hydrolysate in D. bruxellensis.

The influence of nitrate on the physiology of the yeast Dekkera bruxellensis grown under oxygen limitation.

A previous study showed that the use of nitrate by Dekkera bruxellensis might be an advantageous trait when ammonium is limited in sugarcane substrate for ethanol fermentation. The aim of the present work was to evaluate the influence of nitrate on the yeast physiology during cell growth in different carbon sources under oxygen limitation. If nitrate was the sole source of nitrogen, D. bruxellensis cells presented slower growth, diminished sugar consumption and growth-associated ethanol production, when compared to ammonium. These results were corroborated by the increased expression of genes involved in the pentose phosphate (PP) pathway, the tricarboxylic acid (TCA) cycle and ATP synthesis. The presence of ammonium in the mixed medium restored most parameters to the standard conditions. This work may open up a line of investigation to establish the connection between nitrate assimilation and energetic metabolism in D. bruxellensis and their influence on its fermentative capacity in oxygen-limited or oxygen-depleted conditions.

Physiology and gene expression profiles of Dekkera bruxellensis in response to carbon and nitrogen availability.

The assimilation of nitrate, a nitrogenous compound, was previously described as an important factor favoring Dekkera bruxellensis in the competition with Saccharomyces cerevisiae for the industrial sugarcane substrate. In this substrate, nitrogen sources are limited and diverse, and a recent report showed that amino acids enable D. bruxellensis to grow anaerobically. Thus, understanding the regulation of nitrogen metabolism is one fundamental aspect to comprehend the competiveness of D. bruxellensis in the fermentation environment. In the present study, we evaluated the physiological and transcriptional profiles of D. bruxellensis in response to different carbon and nitrogen supplies to determine their influence on growth, sugar consumption, and ethanol production. Besides, the expression of genes coding for nitrogen permeases and enzymes involved in the biosynthesis of glutamate and energetic metabolism were investigated under these conditions. Our data revealed that genes related to nitrogen uptake in D. bruxellensis are under the control of nitrogen catabolite repression. Moreover, we provide indications that glutamate dehydrogenase and glutamate synthase may switch roles as the major pathway for glutamate biosynthesis in D. bruxellensis. Finally, our data showed that in nonoptimal growth conditions, D. bruxellensis leans toward the respiratory metabolism. The results presented herein show that D. bruxellensis and S. cerevisiae share similar regulation of GDH­GOGAT pathway, while D. bruxellensis converts less glucose to ethanol than S. cerevisiae do when nitrogen is limited. The consequence of this particularity to the industrial process is discussed.

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

BACKGROUND: Aquaculture 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. RESULTS: The 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. CONCLUSIONS: Fossil 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.

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

BACKGROUND: Lipid 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. RESULTS: We 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 ß-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. CONCLUSIONS: We suspect that the physiological reason for the accelerated glycerol assimilation and faster lipid production, was primarily the activation of enzymes that provide energy.

Production of short-chain fatty acids (SCFAs) as chemicals or substrates for microbes to obtain biochemicals.

Carboxylic acids have become interesting platform molecules in the last years due to their versatility to act as carbon sources for different microorganisms or as precursors for the chemical industry. Among carboxylic acids, short-chain fatty acids (SCFAs) such as acetic, propionic, butyric, valeric, and caproic acids can be biotechnologically produced in an anaerobic fermentation process from lignocellulose or other organic wastes of agricultural, industrial, or municipal origin. The biosynthesis of SCFAs is advantageous compared to chemical synthesis, since the latter relies on fossil-derived raw materials, expensive and toxic catalysts and harsh process conditions. This review article gives an overview on biosynthesis of SCFAs from complex waste products. Different applications of SCFAs are explored and how these acids can be considered as a source of bioproducts, aiming at the development of a circular economy. The use of SCFAs as platform molecules requires adequate concentration and separation processes that are also addressed in this review. Various microorganisms such as bacteria or oleaginous yeasts can efficiently use SCFA mixtures derived from anaerobic fermentation, an attribute that can be exploited in microbial electrolytic cells or to produce biopolymers such as microbial oils or polyhydroxyalkanoates. Promising technologies for the microbial conversion of SCFAs into bioproducts are outlined with recent examples, highlighting SCFAs as interesting platform molecules for the development of future bioeconomy.