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1.
This study investigated the possibility of utilizing detoxified sugarcane bagasse hydrolysate (DSCBH) as an alternative carbon source to culture Yarrowia lipolytica Po1g for microbial oil and biodiesel production. Sugarcane bagasse hydrolysis with 2.5% HCl resulted in maximum total sugar concentration (21.38 g/L) in which 13.59 g/L is xylose, 3.98 g/L is glucose, and 2.78 g/L is arabinose. Detoxification of SCBH by Ca(OH)2 neutralization reduced the concentration of 5-hydroxymethylfurfural and furfural by 21.31% and 24.84%, respectively. Growth of Y. lipolytica Po1g in DSCBH with peptone as the nitrogen source gave maximum biomass concentration (11.42 g/L) compared to NH4NO3 (6.49 g/L). With peptone as the nitrogen source, DSCBH resulted in better biomass concentration than d-glucose (10.19 g/L), d-xylose (9.89 g/L) and NDSCBH (5.88 g/L). The maximum lipid content, lipid yield and lipid productivity of Y. lipolytica Po1g grown in DSCBH and peptone was 58.5%, 6.68 g/L and 1.76 g/L-day, respectively.  相似文献   

2.
Simultaneous production of citric acid (CA) and invertase by Yarrowia lipolytica A-101-B56-5 (SUC+ clone) growing from sucrose, mixture of glucose and fructose, glucose or glycerol was investigated. Among the tested substrates the highest concentration of CA was reached from glycerol (57.15 g/L) with high yield (YCA/S = 0.6 g/g). When sucrose was used, comparable amount of CA was secreted (45 g/L) with slightly higher yield (YCA/S = 0.643 g/g). In all cultures amount of isocitrate (ICA) was below 2% of total citrates. Considering invertase production, the best carbon source appeared to be sucrose (72 380 U/L). The highest yield of CA and invertase biosynthesis calculated for 1 g of biomass was obtained for cells growing from glycerol (9.9 g/g and 4325 U/g, respectively). Concentrates of extra- and intracellular invertase of the highest activity were obtained from sucrose as substrate (0.5 and 1.8 × 106 U/L, respectively).  相似文献   

3.
In the present report, the effect of glucose and stearin (substrate composed by saturated free-fatty acids) on the production of biomass, reserve lipid, and citric acid by Yarrowia lipolytica ACA-DC 50109 was investigated in nitrogen-limited cultures. Numerical models that were used in order to quantify the kinetic behavior of the above Yarrowia lipolytica strain showed successful simulation, while the optimized parameter values were similar to those experimentally measured and the predictive ability of the models was satisfactory. In nitrogen-limited cultures in which glucose was used as the sole substrate, satisfactory growth and no glucose inhibition occurred, although in some cases the initial concentration of glucose was significantly high (150 g/l). Citric acid production was observed in all trials, which was in some cases notable (final concentration 42.9 g/l, yield 0.56 g per g of sugar consumed). The concentration of unsaturated cellular fatty acids was slightly lower when the quantity of sugar in the medium was elevated. In the cases in which stearin and glucose were used as co-substrates, in spite of the fact that the quantity of cellular lipid inside the yeast cells varied remarkably (from 0.3 to 2.0 g/l – 4 to 20% wt/wt), de novo fatty acid biosynthesis was observed. This activity increased when the yeast cells assimilated higher sugar quantities. The citric acid produced was mainly derived from the catabolism of sugar. Nevertheless, citric acid yield on sugar consumed and citrate specific production rate, as evaluated by the numerical model, presented substantially higher values in the fermentation in which no fat was used as glucose co-substrate compared with the cultures with stearin used as co-substrate.  相似文献   

4.
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.  相似文献   

5.
Yu X  Zheng Y  Dorgan KM  Chen S 《Bioresource technology》2011,102(10):6134-6140
This paper explores the use of the hydrolysate from the dilute sulfuric acid pretreatment of wheat straw for microbial oil production. The resulting hydrolysate was composed of pentoses (24.3 g/L) and hexoses (4.9 g/L), along with some other degradation products, such as acetic acid, furfural, and hydroxymethylfurfural (HMF). Five oleaginous yeast strains, Cryptococcus curvatus, Rhodotorula glutinis, Rhodosporidium toruloides, Lipomyces starkeyi, and Yarrowia lipolytica, were evaluated by using this hydrolysate as substrates. The results showed that all of these strains could use the detoxified hydrolysate to produce lipids while except R. toruloides non-detoxified hydrolysate could also be used for the growth of all of the selective yeast strains. C. curvatus showed the highest lipid concentrations in medium on both the detoxified (4.2 g/L) and non-detoxified (5.8 g/L) hydrolysates. And the inhibitory effect studies on C. curvatus indicated HMF had insignificant impacts at a concentration of up to 3 g/L while furfural inhibited cell growth and lipid content by 72.0% and 62.0% at 1 g/L, respectively. Our work demonstrates that lipid production is a promising alternative to utilize hemicellulosic sugars obtained during pretreatment of lignocellulosic materials.  相似文献   

6.
During cultivation under nitrogen starvation, Yarrowia lipolytica produces a mixture of citric acid and isocitric acid whose ratio is mainly determined by the carbon source used. We report that mitochondrial succinate–fumarate carrier YlSfc1 controls isocitric acid efflux from mitochondria. YlSfc1 purified and reconstituted into liposomes transports succinate, fumarate, oxaloacetate, isocitrate and α-ketoglutarate. YlSFC1 overexpression determined the inversion of isocitric acid/citric acid ratio towards isocitric acid, resulting in 33.4 ± 1.9 g/L and 43.3 ± 2.8 g/L of ICA production in test-tube cultivation with glucose and glycerol, respectively. These titers represent a 4.0 and 6.3-fold increase compared to the wild type. YlSFC1 gene expression was repressed in the wild type strain grown in glucose-based medium compared to olive oil medium explaining the reason for the preferred citric acid production during Y. lipolytica growth on carbohydrates. Coexpression of YlSFC1 and adenosine monophosphate deaminase YlAMPD genes together with inactivation of citrate mitochondrial carrier YlYHM2 gene enhanced isocitric acid accumulation up to 41.4 ± 4.1 g/L with an isocitric acid/citric acid ratio of 14.3 in a small-scale cultivation with glucose as a carbon source. During large-scale cultivation with glucose pulse-feeding, the engineered strain produced 136.7 ± 2.5 g/L of ICA with a process selectivity of 88.1%, the highest reported titer and selectivity to date. These results represent the first reported isocitric acid secretion by Y. lipolytica as a main organic acid during cultivation on carbohydrate. Moreover, we demonstrate for the first time that the replacement of one mitochondrial transport system for another can be an efficient tool for switching product accumulation.  相似文献   

7.
Hu ZC  Zheng YG  Shen YC 《Bioresource technology》2011,102(14):7177-7182
1,3-Dihydroxyacetone can be produced by biotransformation of glycerol with glycerol dehydrogenase from Gluconobacter oxydans cells. Firstly, improvement the activity of glycerol dehydrogenase was carried out by medium optimization. The optimal medium for cell cultivation was composed of 5.6 g/l yeast extract, 4.7 g/l glycerol, 42.1 g/l mannitol, 0.5 g/l K2HPO4, 0.5 g/l KH2PO4, 0.1 g/l MgSO4·7H2O, and 2.0 g/l CaCO3 with the initial pH of 4.9. Secondly, an internal loop airlift bioreactor was applied for DHA production from glycerol by resting cells of G. oxydans ZJB09113. Furthermore, the effects of pH, aeration rate and cell content on DHA production and glycerol feeding strategy were investigated. 156.3 ± 7.8 g/l of maximal DHA concentration with 89.8 ± 2.4% of conversion rate of glycerol to DHA was achieved after 72 h of biotransformation using 10 g/l resting cells at 30 °C, pH 5.0 and 1.5 vvm of aeration rate.  相似文献   

8.
The role of citrate as a physiological modulator of mammalian acetyl-CoA carboxylases (ACCs) has been well studied; however, the mechanism has not been clearly defined. In the current study, we found that citrate activated recombinant human ACC2 by more than ∼1000-fold, but activated recombinant human ACC1 only by ∼4-fold. The data fit best to a model which accounts for cooperative binding of two citrate molecules. Citrate activates ACCs at lower concentrations and inhibits at higher concentrations with apparent Kd values of 0.8 ± 0.3 and 3.4 ± 0.6 mM, and apparent Ki values of 20 ± 8 and 38  ± 8 mM for ACC1 and ACC2, respectively. In the absence of added citrate, both ACC1 and ACC2 were inactivated by avidin rapidly and completely. Addition of 10 mM citrate protected ACC2 from avidin inactivation; however, protection by citrate was less pronounced for ACC1. In response to citrate treatment, different aggregation patterns for the two isoforms were also observed by dynamic light scattering. Although formation of aggregates by both isoforms was sensitive to citrate, with Mg2+ and Mg-citrate addition only formation of the ACC2 aggregates showed a dependence on citrate concentration.Mass spectrometry data indicated phosphorylation of Ser79 of ACC1 (a serine known to regulate activity), and the corresponding Ser221 of ACC2. Taken together, these data suggest that recombinant human ACC1 and ACC2 are differentially activated by citrate, most likely through conformational changes leading to aggregation, with ACC2 being more sensitive to this activator.  相似文献   

9.
Summary Cryptococcus albidus var. albidus CBS 4517 was able to accumulate lipid under nitrogen-limited as well as excess-nitrogen conditions. The highest lipid-producting capacity was, however, observed in nitrogen-limited cultivations. In nitrogen-limited batch cultures, a lipid content of 34% (w/w) in biomass and a maximum specific lipid productivity of 37 mg lipid/g lipid-free biomass·h, was determined. The yield of lipid from glucose was about 0.15 g/g in nitrogen-limited and 0.11 g/g in excess-nitrogen cultures.In a nitrogen-limited fed-batch culture, 12.4 g/l lipid was produced at 90 h of cultivation and the cells contained 46.3% (w/w) lipid.Higher lipid yield and cellular lipid content were observed when inorganic nitrogen sources were used compared with organic. The choice of carbon source was seen to influence growth as well as lipid production and the highest yields of lipid were obtained when glucose, maltose or mannitol was used.A cultivation temperature of 20°C provided the highest lipid productivity compared to 25°C and 30°C. Addition of citrate to the growth medium was seen to have a stimulating effect on the specific lipid productivity.  相似文献   

10.
In this study, crude glycerol with high potassium concentration was purified using acid treatment and used as carbon source for lipid production using Yarrowia lipolytica SKY7. The crude glycerol was purified using phosphoric acid (pH 2) followed by centrifugation. When purified glycerol was used as carbon source for fermentation, higher biomass productivity (0.54 g/L/h) and lipid productivity (0.2 g/L/h) was observed at 96 h compared to crude glycerol. Results indicated that 6.32 g/L potassium in crude glycerol medium was inhibitory for cell growth and lipid production by Y. lipolytica. Yield coefficients, productivities and specific growth rates were calculated for each glycerol medium. The process performance with purified glycerol medium was comparable to that of pure glycerol medium. A higher lipid yield was obtained in purified glycerol medium (0.21 g/g glycerol) than crude glycerol medium (0.124 g/g glycerol). During purification of crude glycerol, KH2PO4 was also produced as by-product. This study provides a way for valorization of crude glycerol with high potassium concentration for microbial lipid production.  相似文献   

11.
Crude glycerol is a major byproduct of the biodiesel industry; previous research has proved the feasibility of producing docosahexaenoic acid (DHA, 22:6 n − 3) through fermentation of the algae Schizochytrium limacinum on crude glycerol. The objective of this work is to investigate the cell growth kinetics, substrate utilization efficiency, and DHA production of the algae through a continuous culture. Steady-state biomass yield, biomass productivity, growth yield on glycerol, specific glycerol consumption rate, and fatty acid composition were investigated within the range of dilution rate (D) from 0.2 to 0.6 day−1, and the range of feed crude glycerol concentration (S0) from 15 to 120 g/L. The maximum specific growth rate was determined as 0.692 day−1. The cells had a true growth yield of 0.283 g/g but with a relatively high maintenance coefficient (0.2216 day−1). The highest biomass productivity of 3.88 g/L-day was obtained at D = 0.3 day−1 and S0 = 60 g/L, while the highest DHA productivity (0.52 g/L-day) was obtained at D = 0.3 day−1 and S0 = 90 g/L due to the higher DHA content at S0 = 90 g/L. The biomass and DHA productivity of the continuous culture was comparable to those of batch culture, while lower than the fed-batch culture, mainly because of the lower DHA content obtained by the continuous culture. Overall, the results show that continuous culture is a powerful tool to investigate the cell growth kinetics and physiological behaviors of the algae growing on biodiesel-derived crude glycerol.  相似文献   

12.
Endophytic fungi (Fusarium mairei) culture broth (EFCB) was added to cell suspension cultures of Taxus cuspidata. After 5 days, cultures of T. cuspidata given 4 ml of EFCB produced a maximal yield of 6.11 mg/l paclitaxel, with a release ratio of 75%, 2- and 6.8-fold, respectively, greater than the controls. The active element in EFCB is an exopolysaccharide of ∼79 kD. Endophytic fungi produced 0.19 mg/l of paclitaxel in its producing medium. However, when the supernatant of Taxus cell suspension cultures from day 20 was added to the paclitaxel-producing medium, the biomass of fungi decreased by 24% and the yield of paclitaxel by 45%. In a co-culture system of plant and fungus, the yield of paclitaxel (12.8 mg/l) was >2-fold higher than that in the EFCB-treatment system.  相似文献   

13.
This study examined the potential of producing an edible fungus, Rhizopus microsporus var. oligosporus, on biodiesel-derived crude glycerol. Prolific fungal growth was observed with a fungal biomass yield of 0.83 ± 0.02 (g biomass increase/g initial biomass) under optimal cultivation conditions (e.g. nonsterile crude glycerol at a concentration of 75% (w/v) with nutrient supplementation and without pH control). The potential of utilizing front-end processed banagrass (Pennisetum purpureum) juice as a source of nutrients for crude glycerol fermentation was evaluated with a 2.3-fold improvement in the fungal biomass yield. The glycerol-derived fungal biomass showed high amounts of threonine, one of the main limiting amino acids in non-ruminant feeds. An inexpensive fungal protein has the potential to reduce meat product prices by lowering the production costs of animal feeds. The application of fungal technology thus provides a unique sustainable option for biodiesel refineries by providing an additional source of revenue from fungal products.  相似文献   

14.
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.  相似文献   

15.
A gene (axe) encoding the AXE thermostable esterase in Thermobifida fusca NTU22 was cloned into a Yarrowia lipolytica P01g host strain. Recombinant expression resulted in extracellular esterase production at levels as high as 70.94 U/ml in Hinton flask culture broth, approximately 140 times higher than observed in a Pichia pastoris expression system. After 72 h of fermentation by the Y. lipolytica transformant in the fed-batch fermentor, the fermentation broth accumulated 41.11 U/ml esterase activity. Rice bran, wheat bran, bagasse and corncob were used as hydrolysis substrates for the esterase, with corncob giving the best ferulic acid yield. The corncob was incubated with T. fusca xylanase (Tfx) for 12 h and then with the AXE esterase for an additional 12 h. Ferulic acid accumulated to 396 μM in the culture broth, a higher concentration than with esterase alone or with Tfx and esterase together for 24 h.  相似文献   

16.
Oxygen sensitivity of hydrogenase is a critical issue in efficient biological hydrogen production. In the present study, oxygen-tolerant [NiFe]-hydrogenase from the marine bacterium, Hydrogenovibrio marinus, was heterologously expressed in Escherichia coli, for the first time. Recombinant E. coli BL21 expressing H. marinus [NiFe]-hydrogenase actively produced hydrogen, but the parent strain did not. Recombinant H. marinus hydrogenase required both nickel and iron for biological activity. Compared to the recombinant E. coli [NiFe]-hydrogenase 1 described in our previous report, recombinant H. marinus [NiFe]-hydrogenase displayed 1.6- to 1.7-fold higher hydrogen production activity in vitro. Importantly, H. marinus [NiFe]-hydrogenase exhibited relatively good oxygen tolerance in analyses involving changes of surface aeration and oxygen proportion within a gas mixture. Specifically, recombinant H. marinus [NiFe]-hydrogenase produced ∼7- to 9-fold more hydrogen than did E. coli [NiFe]-hydrogenase 1 in a gaseous environment containing 5-10% (v/v) oxygen. In addition, purified H. marinus [NiFe]-hydrogenase displayed a hydrogen evolution activity of ∼28.8 nmol H2/(min mg protein) under normal aerobic purification conditions. Based on these results, we suggest that oxygen-tolerant H. marinus [NiFe]-hydrogenase can be employed for in vivo and in vitro biohydrogen production without requirement for strictly anaerobic facilities.  相似文献   

17.
Three Yarrowia lipolytica cell wall proteins (YlPir, YlCWP1 and YlCBM) were evaluated for their ability to display the xylanase TxXYN from Thermobacillus xylanilyticus on the cell surface of Y. lipolytica. The fusion proteins were produced in Y. lipolytica JMY1212, a strain engineered for mono-copy chromosomal insertion, and enabling accurate comparison of anchoring systems. The construction using YlPir enabled cell bound xylanase activity to be maximised (71.6 U/g). Although 48% of the activity was released in the supernatant, probably due to proteolysis at the fusion zone, this system is three times more efficient for the anchoring of TxXYN than the YlCWP1 system formerly developed for Y. lipolytica. As far as we know it represents the best displayed xylanase activity ever published. It could be an attractive alternative anchoring system to display enzymes in Y. lipolytica.  相似文献   

18.
Previously, using γ-irradiation treatment, we isolated a mutant strain of Klebsiella pneumoniae (named GEM167) that showed high-level ethanol production from glycerol. In the present study, in an effort to enhance ethanol production, we used a deletion of the lactate dehydrogenase gene to engineer a mutant strain incapable of lactate synthesis. In the ΔldhA mutant of GEM167, the production of ethanol was significantly increased from 21.5 g/l to 28.9 g/l and from 0.93 g/(l h) to 1.2 g/(l h). Introduction of the Zymomonas mobilis pdc and adhII genes encoding pyruvate decarboxylase and aldehyde dehydrogenase, respectively, further improved the ethanol production level from glycerol to 31.0 g/l; this is the highest level reported to date.  相似文献   

19.
Liu Y  Koh CM  Ji L 《Bioresource technology》2011,102(4):3927-3933
Ustilago maydis is known to produce glycolipid-type biosurfactants. Here, we show that U. maydis is able to efficiently convert biodiesel-derived crude glycerol to glycolipids. We have optimized the medium composition and environmental factors for bioconversion of crude glycerol to glycolipids. The synthetic medium (MinCG) contains 50 g L−1 crude glycerol and 20.3 mg L−1 ammonium citrate as the carbon and nitrogen sources, respectively. The supplementation of trace amount of amino acids, Group-B vitamins and precursors of glycolipids, mannose and erythritol, also improved the final yield. At pH 4.0 and 30 °C, 32.1 g L−1 total glycolipids was produced in a 8.2-day fed-batch bioprocess. Methanol at 2% or above severely inhibited cell growth and production of glycolipids. Our results suggest that U. maydis is an excellent host for the bioconversion of crude glycerol to value-added products.  相似文献   

20.

Background

Malaria is a devastating disease and Plasmodium falciparum is the most lethal parasite infecting humans. Understanding the biology of this parasite is vital in identifying potential novel drug targets. During every 48-hour intra-erythrocytic asexual replication cycle, a single parasite can produce up to 32 progeny. This extensive proliferation implies that parasites require substantial amounts of lipid precursors for membrane biogenesis. Glycerol kinase is a highly conserved enzyme that functions at the interface of lipid synthesis and carbohydrate metabolism. P. falciparum glycerol kinase catalyzes the ATP-dependent phosphorylation of glycerol to glycerol-3-phosphate, a major phospholipid precursor.

Methods

The P. falciparum glycerol kinase gene was disrupted using double crossover homologous DNA recombination to generate a knockout parasite line. Southern hybridization and mRNA analysis were used to verify gene disruption. Parasite growth rates were monitored by flow cytometry. Radiolabelling studies were used to assess incorporation of glycerol into parasite phospholipids.

Results

Disruption of the P. falciparum glycerol kinase gene produced viable parasites, but their growth was significantly reduced to 56.5 ± 1.8% when compared to wild type parasites. 14C-glycerol incorporation into the major phospholipids of the parasite membrane, phosphatidylcholine and phosphatidylethanolamine, was 48.4 ± 10.8% and 53.1 ± 5.7% relative to an equivalent number of wild type parasites.

Conclusions

P. falciparum glycerol kinase is required for optimal intra-erythrocytic asexual parasite development. Exogenous glycerol may be used as an alternative carbon source for P. falciparum phospholipid biogenesis, despite the lack of glycerol kinase to generate glycerol-3-phosphate.

General significance

These studies provide new insight into glycerolipid metabolism in P. falciparum.  相似文献   

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