Succinic acid production from n‐alkanes

A novel process of production of succinic acid (SA) has been developed, which includes the synthesis of alpha‐ketoglutaric acid by a thiamine‐auxotrophic yeast strain Yarrowia lipolytica VKM Y‐2412 from n‐alkanes and subsequent oxidation of the acid by hydrogen peroxide to SA. The concentration of SA in the culture broth and its yield were found to be 38.8 g/L and 82.45% of n‐alkane consumed, respectively. The isolation procedure involved the extraction of the residual alkanes with the mixture of ethyl acetate and hexane, the decomposition of H₂O₂ in the filtrate followed by filtrate bleaching and acidification with a mineral acid; the evaporation of filtrate and the ethanol extraction of SA from lyophilized residue. The purity of the SA isolated from the culture liquid filtrate reached 99.5%.     

Citric acid production from sucrose by recombinant Yarrowia lipolytica using semicontinuous fermentation

The genetically modified yeast strain Yarrowia lipolytica H222‐S4(p67ICL1)T5 is able to utilize sucrose as a carbon source and to produce citric and isocitric acids in a more advantageous ratio as compared to its wild‐type equivalent. In this study, the effect of pH of the fermentation broth (pH 6.0 and 7.0) and proteose‐peptone addition on citric acid production by the recombinant yeast strain were investigated. It was found that the highest citric acid production occurred at pH 7.0 without any addition of proteose‐peptone. Furthermore, two process strategies (fed‐batch and repeated fed‐batch) were tested for their applicability for use in citric acid production from sucrose by Y. lipolytica. Repeated fed‐batch cultivation was found to be the most effective process strategy: in 3 days of cycle duration, approximately 80 g/L citric acid was produced, the yield was at least 0.57 g/g and the productivity was as much as 1.1 g/Lh. The selectivity of the bioprocess for citric acid was always higher than 90% from the very beginning of the fermentation due to the genetic modification, reaching values of up to 96.4% after 5 days of cycle duration.     

High‐yield lipid production from lignocellulosic biomass using engineered xylose‐utilizing Yarrowia lipolytica

Lignocellulosic biomass shows high potential as a renewable feedstock for use in biodiesel production via microbial fermentation. Yarrowia lipolytica, an emerging oleaginous yeast, has been engineered to efficiently convert xylose, the second most abundant sugar in lignocellulosic biomass, into lipids for lignocellulosic biodiesel production. Yet, the lipid yield from xylose or lignocellulosic biomass remains far lower than that from glucose. Here we developed an efficient xylose‐utilizing Y. lipolytica strain, expressing an isomerase‐based pathway, to achieve high‐yield lipid production from lignocellulosic biomass. The newly developed xylose‐utilizing Y. lipolytica, YSXID, produced 12.01 g/L lipids with a maximum yield of 0.16 g/g, the highest ever reported, from lignocellulosic hydrolysates. Consequently, this study shows the potential of isomerase‐based xylose‐utilizing Y. lipolytica for economical and sustainable production of biodiesel and oleochemicals from lignocellulosic biomass.     

Biotechnological conversions of bio‐diesel‐derived crude glycerol by Yarrowia lipolytica strains

In the present report, crude glycerol, waste discharged from bio‐diesel production, was used as carbon substrate for three natural Yarrowia lipolytica strains (LFMB 19, LFMB 20 and ACA‐YC 5033) during growth in nitrogen‐limited submerged shake‐flask experiments. In media with initial glycerol concentration of 30 g/L, all strains presented satisfactory microbial growth and complete glycerol uptake. Although culture conditions favored the secretion of citric acid (and potentially the accumulation of storage lipid), for the strains LFMB 19 and LFMB 20, polyol mannitol was the principal metabolic product synthesized (maximum quantity 6.0 g/L, yield 0.20–0.26 g per g of glycerol consumed). The above strains produced small quantities of lipids and citric acid. In contrast, Y. lipolytica ACA‐YC 5033 produced simultaneously higher quantities of lipid and citric acid and was further grown on crude glycerol in nitrogen‐limited experiments, with constant nitrogen and increasing glycerol concentrations (70–120 g/L). Citric acid and lipid concentrations increased with increment of glycerol; maximum total citric acid 50.1 g/L was produced (yield 0.44 g per g of glycerol) while simultaneously 2.0 g/L of fat were accumulated inside the cells (0.31 g of lipid per g of dry weight). Cellular lipids were mainly composed of neutral fraction, the concentration of which substantially increased with time. Moreover, in any case, the phospholipid fraction was more unsaturated compared with total and neutral lipids, while at the early growth step, microbial lipid was more rich in saturated fatty acids (e.g. C16:0 and C18:0) compared with the stationary phase.     

Enhanced solid‐state citric acid bio‐production using apple pomace waste through surface response methodology

Aims: To evaluate the potential of apple pomace (AP) supplemented with rice husk for hyper citric acid production through solid‐state fermentation by Aspergillus niger NRRL‐567. Optimization of two key parameters, such as moisture content and inducer (ethanol and methanol) concentration was carried out by response surface methodology. Methods and Results: In this study, the effect of two crucial process parameters for solid‐state citric acid fermentation by A. niger using AP waste supplemented with rice husk were thoroughly investigated in Erlenmeyer flasks through response surface methodology. Moisture and methanol had significant positive effect on citric acid production by A. niger grown on AP (P < 0·05). Higher values of citric acid on AP by A. niger (342·41 g kg^?1 and 248·42 g kg^?1 dry substrate) were obtained with 75% (v/w) moisture along with two inducers [3% (v/w) methanol and 3% (v/w) ethanol] with fermentation efficiency of 93·90% and 66·42%, respectively depending upon the total carbon utilized after 144 h of incubation period. With the same optimized parameters, conventional tray fermentation was conducted. The citric acid concentration of 187·96 g kg^?1 dry substrate with 3% (v/w) ethanol and 303·34 g kg^?1 dry substrate with 3% (v/w) methanol were achieved representing fermentation efficiency of 50·80% and 82·89% in tray fermentation depending upon carbon utilization after 120 h of incubation period. Conclusions: Apple pomace proved to be the promising substrate for the hyper production of citric acid through solid‐state tray fermentation, which is an economical technique and does not require any sophisticated instrumentation. Significance and Impact of the Study: The study established that the utilization of agro‐industrial wastes have positive repercussions on the economy and will help to meet the increasing demands of citric acid and moreover will help to alleviate the environmental problems resulting from the disposal of agro‐industrial wastes.     

Bioengineering triacetic acid lactone production in Yarrowia lipolytica for pogostone synthesis

Yarrowia lipolytica is an oleaginous yeast that is recognized for its ability to accumulate high levels of lipids, which can serve as precursors to biobased fuels and chemicals. Polyketides, such as triacetic acid lactone (TAL), can also serve as a precursor for diverse commodity chemicals. This study used Y. lipolytica as a host organism for the production of TAL via expression of the 2‐pyrone synthase gene from Gerbera hybrida. Induction of lipid biosynthesis by nitrogen‐limited growth conditions increased TAL titers. We also manipulated basal levels of TAL production using a DNA cut‐and‐paste transposon to mobilize and integrate multiple copies of the 2‐pyrone synthase gene. Strain modifications and batch fermentation in nitrogen‐limited medium yielded TAL titers of 2.6 g/L. Furthermore, we show that minimal medium allows TAL to be readily concentrated at >94% purity and converted at 96% yield to pogostone, a valuable antibiotic. Modifications of this reaction scheme yielded diverse related compounds. Thus, oleaginous organisms have the potential to be flexible microbial biofactories capable of economical synthesis of platform chemicals and the generation of industrially relevant molecules.     

Biosensor online control of citric acid production from glucose by Yarrowia lipolytica using semicontinuous fermentation

Our study aimed at the development of an effective method for citric acid production from glucose by use of the yeast Yarrowia lipolytica. The new method included an automated bioprocess control using a glucose biosensor. Several fermentation methodologies including batch, fed‐batch, repeated batch and repeated fed‐batch cultivation were tested. The best results were achieved during repeated fed‐batch cultivation: Within 3 days of cycle duration, approximately 100 g/L citric acid were produced. The yields reached values between 0.51 and 0.65 g/g and the selectivity of the bioprocess for citric acid was as high as 94%. Due to the elongation of the production phase of the bioprocess with growth‐decoupled citric acid production, and by operating the fermentation in cycles, an increase in citric acid production of 32% was achieved compared with simple batch fermentation.     

Aconitase overexpression changes the product ratio of citric acid production by <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis>

The yeast Yarrowia lipolytica secretes high amounts of various organic acids, like citric acid (CA) and isocitric acid (ICA) under an excess of carbon source and several conditions of growth limitation. Depending on the carbon source used, Y. lipolytica strains produce a mixture of CA and ICA in a characteristic ratio. To examine whether this CA/ICA product ratio can be influenced by gene–dose-dependent overexpression of aconitase (ACO)-encoding gene ACO1, a recombinant Y. lipolytica strain was constructed containing multiple copies of ACO1. The high-level expression of ACO in the ACO1 multicopy integrative transformant resulted in a shift of the CA/ICA product pattern into the direction of ICA. On sunflower oil, a striking increase of the ICA proportion from 35–49% to 66–71% was observed compared to wild-type strains without influencing the total amount of acids (CA and ICA) produced. On glycerol, glucose or sucrose, the ICA proportion increased only moderately from 10–12% to 13–17%. This moderate shift into the direction of ICA was also observed in an icl1-defective strain.     

Lipid production by yeasts grown on crude glycerol from biodiesel industry

The main carbon source used for growth by four yeast strains (Yarrowia lipolytica CCMA 0357, Y. lipolytica CCMA 0242, Wickerhamomyces anomalus CCMA 0358, and Cryptococcus humicola CCMA 0346) and their lipid production were evaluated, using different concentrations of crude and pure glycerol and glucose. Whereas crude glycerol (100?g/L) was the main carbon source used by Y. lipolytica CCMA 0357 (nearly 15?g/L consumed at 120?hr) and W. anomalus CCMA 0358 (nearly 45.10?g/L consumed at 48?hr), pure glycerol (150?g/L) was the main one used by C. humicola CCMA 0346 (nearly 130?g/L consumed). On the other hand, Y. lipolytica CCMA 0242 used glucose (100?g/L) as its main source of carbon (nearly 96.48?g/L consumed). Y. lipolytica CCMA 0357 demonstrated the highest lipid production [about 70% (wt/wt)], forming palmitic (45.73% of fatty acid composition), stearic (16.43%), palmitoleic (13.29%), linolenic (10.77%), heptadecanoic (4.07%), and linoleic (14.14%) acids. Linoleic acid, an essential fatty acid, was produced by all four yeast strains but in varying degrees, representing 70.42% of the fatty acid profile of lipids produced by C. humicola CCMA 0346.     

Production of added‐value metabolites by Yarrowia lipolytica growing in olive mill wastewater‐based media under aseptic and non‐aseptic conditions

Yarrowia lipolytica ACA‐YC 5033 was grown on glucose‐based media in which high amounts of olive mill wastewaters (OMWs) had been added. Besides shake‐flask aseptic cultures, trials were also performed in previously pasteurized media while batch bioreactor experiments were also done. Significant decolorization (～58%) and remarkable removal of phenolic compounds (～51% w/w) occurred, with the latter being amongst the highest ones reported in the international literature, as far as yeasts were concerned during their growth on phenol‐containing media. In nitrogen‐limited flask fermentations the microorganism produced maximum citric acid quantity ≈19.0 g/L [simultaneous yield of citric acid produced per unit of glucose consumed (Y_Cit/Glc)≈0.74 g/g]. Dry cell weight (DCW) values decreased at high phenol‐containing media, but, on the other hand, the addition of OMWs induced reserve lipid accumulation. Maximum citric acid concentration achieved (≈52.0 g/L; Y_Cit/Glc≈0.64 g/g) occurred in OMW‐based high sugar content media (initial glucose added at ≈80.0 g/L). The bioprocess was successfully simulated by a modified logistic growth equation. A satisfactory fitting on the experimental data occurred while the optimized parameter values were found to be similar to those experimentally measured. Finally, a non‐aseptic (previously pasteurized) trial was performed and its comparison with the equivalent aseptic experiment revealed no significant differences. Yarrowia lipolytica hence can be considered as a satisfactory candidate for simultaneous OMWs bioremediation and the production of added‐value compounds useful for the food industry.     

油酸促进灵芝三萜液态深层发酵的工艺研究及规模化验证

灵芝三萜是灵芝中主要的活性成分之一,前期研究发现油酸可以促进灵芝三萜液态深层发酵下的发酵合成。本研究主要对油酸促进灵芝三萜液态深层发酵的工艺进行优化,并进行3L发酵罐规模的验证。通过单因素实验考察油酸的添加方式、添加时间和添加浓度对灵芝三萜的影响,结合响应面实验,获得最优工艺条件并进行验证：在发酵第32h添加1.21%高温灭菌油酸,最高灵芝三萜含量为42.69mg/g;在发酵第7h添加1.35%过滤除菌油酸,最高三萜含量为43.38mg/g,分别比对照提高2.04倍和2.08倍。在1 000mL摇瓶中添加高温灭菌油酸和过滤除菌油酸,灵芝三萜含量分别为32.18和32.48mg/g,为对照的1.96倍和1.95倍;在3L发酵罐规模下灵芝三萜含量分别为28.66和25.13mg/g,为对照的1.62倍和1.42倍。本研究系统优化了油酸促进灵芝三萜液态深层发酵的工艺条件,并在与工业生产相对应的3L发酵罐上进行验证。该研究可为灵芝三萜的规模化发酵提供重要参考和借鉴。     

Microbial production of d‐lactic acid from dried distiller's grains with solubles

d ‐Lactic acid production is gaining increasing attention due to the thermostable properties of its polymer, poly‐d ‐lactic acid . In this study, Lactobacillus coryniformis subsp. torquens, was evaluated for its ability to produce d ‐lactic acid using Dried Distiller's Grains with Solubles (DDGS) hydrolysate as the substrate. DDGS was first subjected to alkaline pretreatment with sodium hydroxide to remove the hemicellulose component and the generated carbohydrate‐rich solids were then subjected to enzymatic hydrolysis using cellulase mixture Accellerase® 1500. When comparing separate hydrolysis and fermentation and simultaneous saccharification and fermentation (SSF) of L. coryniformis on DDGS hydrolysate, the latter method demonstrated higher d ‐lactic acid production (27.9 g/L, 99.9% optical purity of d ‐lactic acid), with a higher glucose to d ‐lactic acid conversion yield (84.5%) compared to the former one (24.1 g/L, 99.9% optical purity of d ‐lactic acid). In addition, the effect of increasing the DDGS concentration in the fermentation system was investigated via a fed‐batch SSF approach, where it was shown that the d ‐lactic acid production increased to 38.1 g/L and the conversion yield decreased to 70%. In conclusion, the SSF approach proved to be an efficient strategy for the production of d ‐lactic acid from DDGS as it reduced the overall processing time and yielded high d ‐lactic acid concentrations.     

Production of polymalic acid and malic acid by Aureobasidium pullulans fermentation and acid hydrolysis

Malic acid is a dicarboxylic acid widely used in the food industry and also a potential C4 platform chemical that can be produced from biomass. However, microbial fermentation for direct malic acid production is limited by low product yield, titer, and productivity due to end‐product inhibition. In this work, a novel process for malic acid production from polymalic acid (PMA) fermentation followed by acid hydrolysis was developed. First, a PMA‐producing Aureobasidium pullulans strain ZX‐10 was screened and isolated. This microbe produced PMA as the major fermentation product at a high‐titer equivalent to 87.6 g/L of malic acid and high‐productivity of 0.61 g/L h in free‐cell fermentation in a stirred‐tank bioreactor. Fed‐batch fermentations with cells immobilized in a fibrous‐bed bioreactor (FBB) achieved the highest product titer of 144.2 g/L and productivity of 0.74 g/L h. The fermentation produced PMA was purified by adsorption with IRA‐900 anion‐exchange resins, achieving a ～100% purity and a high recovery rate of 84%. Pure malic acid was then produced from PMA by hydrolysis with 2 M sulfuric acid at 85°C, which followed the first‐order reaction kinetics. This process provides an efficient and economical way for PMA and malic acid production, and is promising for industrial application. Biotechnol. Bioeng. 2013; 110: 2105–2113. © 2013 Wiley Periodicals, Inc.     

Engineering <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> for poly-3-hydroxybutyrate production

Strains of Yarrowia lipolytica were engineered to express the poly-3-hydroxybutyrate (PHB) biosynthetic pathway. The genes for β-ketothiolase, NADPH-dependent acetoacetyl-CoA reductase, and PHB synthase were cloned and inserted into the chromosome of Y. lipolytica. In shake flasks, the engineered strain accumulated PHB to 1.50 and 3.84% of cell dry weight in complex medium supplemented with glucose and acetate as carbon source, respectively. In fed-batch fermentation using acetate as sole carbon source, 7.35 g/l PHB (10.2% of cell dry weight) was produced. Selection of Y. lipolytica as host for PHB synthesis was motivated by the fact that this organism is a good lipids producer, which suggests robust acetyl-CoA supply also the precursor of the PHB pathway. Acetic acid could be supplied by gas fermentation, anaerobic digestion, and other low-cost supply route.     

Effect of lipids and detergents on the production and solubilization of extracellular lipase inYarrowia lipolytica

Triglycerides, oleic acid but not fatty acid-containing, nonionic detergents (Spans, Tweens) were able to stimulate the synthesis of cell-bound and soluble lipase ofYarrowia lipolytica grown in a complex medium containing citrate and urea. The optimal concentration of olive oil for induction was 0.5% (W/V). The combined effect of a high ionic strength (0.75 mol/L KCl) and of digitonin (2 mmol/L) at pH 7.6 resulted in solubilization of 80% of the cell-bound lipase and a significant activation of the enzyme. Comparison of twoY. lipolytica strains showed the effects of Mg²⁺ and Fe³⁺ concentrations in the medium on the synthesis of the enzyme to be strongly strain-dependent.     

Biochemical and kinetic evaluation of lipase and biosurfactant assisted ex novo synthesis of microbial oil for biodiesel production by Yarrowia lipolytica utilizing chicken tallow

The present study explores the production of biodiesel, a sustainable replacement for depleting fossil fuel by utilizing microbial oil, which was procured from Yarrowia lipolytica employing chicken tallow as the carbon substrate. Chicken tallow, yeast extract, and MgSO₄·7H₂O were screened for biomass production through Plackett–Burman design. Further, Box–Behnken design analysis was performed, and the optimal concentration of the medium variables was found to be 20 g/L of chicken tallow, 7.0 g/L of yeast extract, and 0.45 g/L of MgSO₄·7H₂O.The various parameters viz., pH (6), temperature (30 °C), RPM (150), inoculum volume (5%, v/v), and C/N ratio (100) were optimized for maximal biomass and lipid yield, and lipid content. Nile red-stained cells were observed for intracellular lipid bodies using fluorescence microscopy, and its fluorescence intensity was measured bythe flow cytometer. The dimorphic transition and substrate assimilation of Y. lipolytica were analyzed using scanning electron microscopy (SEM), and Fourier transform infrared spectroscopy (FT-IR). Batch kinetic studies revealed the concomitant synthesis of microbial lipid (4.16 g/L), lipase (43 U/mL), and biosurfactant (1.41 g/L). The GC-MS analysis of microbial oil presented the fatty acid profile as oleic acid (49.15%), palmitic acid (29.83%), stearic acid (11.43%), linoleic acid (3.83%), palmitoleic acid (3.77%), and myristic acid (1.32%).     

New recombinant strains of the yeast <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> with overexpression of the aconitate hydratase gene for the obtainment of isocitric acid from rapeseed oil

The yeast Yarrowia lipolytica is capable of high-intensity synthesis (overproduction) of citric (CA) and isocitric (ICA) acids under nitrogen limitation. The ratio of the synthesized acids depends on the producing strains used and the expression level of the aconitate hydratase gene (ACO1). Recombinant variants with overexpression of the multicopy ACO1 gene have been obtained based on the natural ICA-producing strain Y. lipolytica 672. A recombinant strain Y. lipolytica 20, which has an isocitrate-citrate ratio shifted towards ICA (2.3: 1) as compared to the parental strain (1.1: 1), has been selected. Culturing of the 20 variant in a 10 L reactor has resulted in the production of 72.6 g/L of ICA and 29.0 g/L of CA with a ratio of 2.5: 1. This makes it possible to regard Y. lipolytica 20 as a promising producer for the development of an industrial process for isocitrate production.     

γ-decalactone production by Yarrowia lipolytica and Lindnera saturnus in crude glycerol

Flavor compounds are commonly obtained from chemical synthesis or extracted from plants. These sources have disadvantages, such as racemic mixture generation, more steps to yield the final product, low yield, and high cost, making the microbial fermentation an alternative and potential way to obtain flavor compounds. The most important lactone for flavor application is γ-decalactone, which has an aroma of peach and can be obtained by ricinoleic acid biotransformation through yeast peroxisomal β-oxidation. The aim of this work was to use crude glycerol, a residual biodiesel industry, for the production of bioaroma from two different yeasts. Yarrowia lipolytica CCMA 0357 and Lindnera saturnus CCMA 0243 were grown at different concentrations (10, 20, and 30% w/v) of substrates (castor oil and crude glycerol) for γ-decalactone production. L. saturnus CCMA 0243 produced higher concentration of y-decalactone (5.8?g/L) in crude glycerol, whereas Y. lipolytica CCMA 0357 showed a maximum production in castor oil (3.5?g/L). Crude glycerol showed better results for γ-decalactone production when compared to castor oil. L. saturnus CCMA 0243 has been shown to have a high potential for γ-decalactone production from crude glycerol.