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.

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.

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.

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.

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.

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.

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.

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.

Microbial lipid production from crude glycerol and hemicellulosic hydrolysate with oleaginous yeasts.

BACKGROUND: Crude glycerol (CG) and hemicellulose hydrolysate (HH) are low-value side-products of biodiesel transesterification and pulp-and paper industry or lignocellulosic ethanol production, respectively, which can be converted to microbial lipids by oleaginous yeasts. This study aimed to test the ability of oleaginous yeasts to utilise CG and HH and mixtures of them as carbon source. RESULTS: Eleven out of 27 tested strains of oleaginous yeast species were able to grow in plate tests on CG as sole carbon source. Among them, only one ascomycetous strain, belonging to Lipomyces starkeyi, was identified, the other 10 strains were Rhodotorula spec. When yeasts were cultivated in mixed CG/ HH medium, we observed an activation of glycerol conversion in the Rhodotorula strains, but not in L. starkeyi. Two strains-Rhodotorula toruloides CBS 14 and Rhodotorula glutinis CBS 3044 were further tested in controlled fermentations in bioreactors in different mixtures of CG and HH. The highest measured average biomass and lipid concentration were achieved with R. toruloides in 10% HH medium mixed with 55 g/L CG-19.4 g/L and 10.6 g/L, respectively, with a lipid yield of 0.25 g lipids per consumed g of carbon source. Fatty acid composition was similar to other R. toruloides strains and comparable to that of vegetable oils. CONCLUSIONS: There were big strain differences in the ability to convert CG to lipids, as only few of the tested strains were able to grow. Lipid production rates and yields showed that mixing GC and HH have a stimulating effect on lipid accumulation in R. toruloides and R. glutinis resulting in shortened fermentation time to reach maximum lipid concentration, which provides a new perspective on converting these low-value compounds to microbial lipids.

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.

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.

Assembly and Analysis of the Genome Sequence of the Yeast Brettanomyces naardenensis CBS 7540.

Brettanomyces naardenensis is a spoilage yeast with potential for biotechnological applications for production of innovative beverages with low alcohol content and high attenuation degree. Here, we present the first annotated genome of B. naardenensis CBS 7540. The genome of B. naardenensis CBS 7540 was assembled into 76 contigs, totaling 11,283,072 nucleotides. In total, 5168 protein-coding sequences were annotated. The study provides functional genome annotation, phylogenetic analysis, and discusses genetic determinants behind notable stress tolerance and biotechnological potential of B. naardenensis.

FT-NIR: a tool for rapid intracellular lipid quantification in oleaginous yeasts.

BACKGROUND: Lipid extraction for quantification of fat content in oleaginous yeasts often requires strong acids and harmful organic solvents; it is laborious and time-consuming. Therefore, in most cases just endpoint measurements of lipid accumulation are performed and kinetics of intracellular lipid accumulation is difficult to follow. To address this, we created a prediction model using Fourier-transform near-infrared (FT-NIR) spectroscopy. This method allows to measure lipid content in yeast. METHODS: The FT-NIR calibration sets were constructed from spectra of freeze-dried cells of the oleaginous yeasts Rhodotorula toruloides CBS 14, Lipomyces starkeyi CBS 1807 and Yarrowia lipolytica CBS 6114. The yeast cells were obtained from different cultivation conditions. Freeze-dried cell pellets were scanned using FT-NIR in the Multi Purpose Analyser (MPA) from Bruker. The obtained spectra were assigned corresponding to total fat content, obtained from lipid extraction using a modified Folch method. Quantification models using partial least squares (PLS) regression were built, and the calibration sets were validated on independently cultivated samples. The R. toruloides model was additionally tested on Rhodotorula babjevae DBVPG 8058 and Rhodotorula glutinis CBS 2387. RESULTS: The R 2 of the FT-NIR model for R. toruloides was 98%, and the root mean square error of cross-validation (RMSECV) was 1.53. The model was validated using a separate set of R. toruloides samples with a root mean square error of prediction (RMSEP) of 3.21. The R 2 of the Lipomyces model was 96%, with RMSECV 2.4 and RMSEP 3.8. The R 2 of the mixed model, including all tested yeast strains, was 90.5%, with RMSECV 2.76 and RMSEP 3.22, respectively. The models were verified by predicting the total fat content in newly cultivated and freeze-dried samples. Additionally, the kinetics of lipid accumulation of a culture were followed and compared with standard lipid extraction methods. CONCLUSIONS: Using FT-NIR spectroscopy, we have developed a faster, less laborious and non-destructive quantification of yeast intracellular lipid content compared to methods using lipid extraction.

Biochemical profiling, prediction of total lipid content and fatty acid profile in oleaginous yeasts by FTIR spectroscopy.

BACKGROUND: Oleaginous yeasts are considered as a potential lipid source for food, feed and biofuel production. In order to make the yeast-based lipid production environmentally and economically sustainable, there is a need for screening studies in order to find the best yeast lipid producers on different substrates, and to optimize cultivation conditions. Since the target parameter of such screening studies are lipid amounts and profiles, an analytical technique that is able to perform lipid analyses rapidly, reproducible and with high precision is highly desirable. The main objective of this study was to establish the non-invasive high-throughput Fourier transform infrared (FTIR) spectroscopy analysis for the prediction of lipid content and profile in oleaginous yeasts. RESULTS: High-throughput FTIR spectroscopy allowed characterizing the total biochemical profile of oleaginous yeasts and enabled us to identify strains and substrate(s) providing the highest total lipid content. Some of the yeast strains grown under nitrogen-limiting conditions with glucose/xylose/mixture of glucose and xylose as carbon sources were accumulating lipids with a high proportion of free fatty acids. FTIR spectra were used to predict gravimetric and gas chromatography data by establishing multivariate calibration models. Coefficients of determination (R 2) for calibration models were obtained in a range between 0.62 and 0.92 for predicting lipid content. When using an independent test set, R 2 values between 0.53 and 0.79 were achieved for predicting fatty acid profile. The best spectral region(s) for the prediction of total lipid content was 3100-2800 cm-1 combined with 1800-700 cm-1, and for prediction of summed saturated (SAT), monounsaturated (MUFA) and polyunsaturated (PUFA) fatty acids: 3100-2800 cm-1, 3100-2800 cm-1 combined with 1700-1715 cm-1 and 3100-2800 cm-1 combined with 1800-1715 cm-1, respectively. The highest lipid accumulation was observed for strains Rhodotorula babjevae DBVPG 8058 on glucose and mixture of glucose and xylose and Lipomyces starkeyi CBS 2512 on xylose. CONCLUSIONS: Applying FTIR spectroscopy combined with multivariate data analysis allows performing rapid, non-invasive, reproducible and precise quantitative predictions of total lipid content and lipid profile. It allows also detecting different lipid fractions as triacylglycerols (TAGs) and free fatty acids and evaluating the total biochemical profile of cells. Several yeast strains with high lipid accumulation were identified.

Chromosomal genome assembly of the ethanol production strain CBS 11270 indicates a highly dynamic genome structure in the yeast species Brettanomyces bruxellensis.

Here, we present the genome of the industrial ethanol production strain Brettanomyces bruxellensis CBS 11270. The nuclear genome was found to be diploid, containing four chromosomes with sizes of ranging from 2.2 to 4.0 Mbp. A 75 Kbp mitochondrial genome was also identified. Comparing the homologous chromosomes, we detected that 0.32% of nucleotides were polymorphic, i.e. formed single nucleotide polymorphisms (SNPs), 40.6% of them were found in coding regions (i.e. 0.13% of all nucleotides formed SNPs and were in coding regions). In addition, 8,538 indels were found. The total number of protein coding genes was 4897, of them, 4,284 were annotated on chromosomes; and the mitochondrial genome contained 18 protein coding genes. Additionally, 595 genes, which were annotated, were on contigs not associated with chromosomes. A number of genes was duplicated, most of them as tandem repeats, including a six-gene cluster located on chromosome 3. There were also examples of interchromosomal gene duplications, including a duplication of a six-gene cluster, which was found on both chromosomes 1 and 4. Gene copy number analysis suggested loss of heterozygosity for 372 genes. This may reflect adaptation to relatively harsh but constant conditions of continuous fermentation. Analysis of gene topology showed that most of these losses occurred in clusters of more than one gene, the largest cluster comprising 33 genes. Comparative analysis against the wine isolate CBS 2499 revealed 88,534 SNPs and 8,133 indels. Moreover, when the scaffolds of the CBS 2499 genome assembly were aligned against the chromosomes of CBS 11270, many of them aligned completely, some have chunks aligned to different chromosomes, and some were in fact rearranged. Our findings indicate a highly dynamic genome within the species B. bruxellensis and a tendency towards reduction of gene number in long-term continuous cultivation.

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.

Oleaginous yeast as a component in fish feed.

This study investigates the replacement of vegetable oil (VO) in aquaculture feed for Arctic char (Salvelinus alpinus) with oil produced by the oleaginous yeast Lipomyces starkeyi grown in lignocellulose (wheat straw) hydrolysate. VO is extensively used to partially replace fish oil in aquaculture feed, which can be seen as non-sustainable. VO itself is becoming a limited resource. Plant oils are used in many different applications, including food, feed and biodiesel. Its replacement in non-food applications is desirable. For this purpose, yeast cells containing 43% lipids per g dry weight were mechanically disrupted and incorporated into the fish feed. There were no significant differences in this pilot study, regarding weight and length gain, feed conversion ratio, specific growth rate, condition factor and hepatosomatic index between the control and the yeast oil fed group. Fatty and amino acid composition of diet from both groups was comparable. Our results in fish demonstrate that it is possible to replace VO by yeast oil produced from lignocellulose, which may broaden the range of raw materials for food production and add value to residual products of agriculture and forestry.

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.