Β-carotene Yield Research Articles

Protoplast fusion between Blakeslea trispora 14,271 (+) and 14,272 (-) enhanced the yield of lycopene and β-carotene.

Blakeslea trispora, a heterothallic Zygomycota with two mating types (termed "plus" and "minus"), is an ideal source of lycopene and β-carotene. The lycopene and β-carotene yields when the two type strains are used for fermentation separately are lower than those when they are joint together. To enhance the yield of lycopene and β-carotene in B. trispora, protoplast fusion technology was carried out between ATCC 14,271 (+) and ATCC 14,272 (-). After protoplast preparation, protoplast fusion, fusion sorting, fusion regeneration, and high-throughput screening, two fusions (Fu-1and Fu-2) with high lycopene and β-carotene yields were obtained. The lycopene yields of Fu-1 and Fu-2 were increased to 0.60mg/gDW and 0.90mg/gDW, which were respectively 3.62- and 5.44-fold those of 14,271 and 1.76- and 2.64-fold those of 14,272. The β-carotene yields of Fu-1 and Fu-2 were increased to 22.07mg/gDW and 36.93mg/gDW, which were respectively 1.72- and 2.89-fold those of 14,271 and 1.23- and 2.06-fold those of 14,272. In this study, the protoplast fusion technique was successfully used in Blakeslea trispora, providing new ideas for improving lycopene and β-carotene production.

Production of β-carotene in Saccharomyces cerevisiae through altering yeast lipid metabolism.

Saccharomyces cerevisiae is a widely used cell factory for the production of fuels and chemicals. However, as a non-oleaginous yeast, S. cerevisiae has a limited production capacity for lipophilic compounds, such as β-carotene. To increase its accumulation of β-carotene, we engineered different lipid metabolic pathways in a β-carotene producing strain and investigated the relationship between lipid components and the accumulation of β-carotene. We found that overexpression of sterol ester synthesis genes ARE1 and ARE2 increased β-carotene yield by 1.5-fold. Deletion of phosphatidate phosphatase (PAP) genes (PAH1, DPP1, and LPP1) also increased β-carotene yield by twofold. Combining these two strategies resulted in a 2.4-fold improvement in β-carotene production compared with the starting strain. These results demonstrated that regulating lipid metabolism pathways is important for β-carotene accumulation in S. cerevisiae, and may also shed insights to the accumulation of other lipophilic compounds in yeast.

Natural β-Carotene Production by Blakeslea trispora Cultivated in Spanish-Style Green Olive Processing Wastewaters.

In the current research, the potential of Spanish-style green olive processing wastewaters (lye and washing waters) exploitation toward natural β-carotene production by Blakeslea trispora was tested for the first time. Mating culture generated by the joint cultivation of the heterothallic fungal strains ATCC 14271 and 14272 in the non-sterile lye and washing waters was able to grow, achieving the phytotoxic hydroxytyrosol degradation by 57.3% and 66.8%, respectively. However, the low sugar and nitrogen content of the streams did not favor carotenogenesis. Alternatively, in the nutrient-enriched effluents, a notable quantity of β-carotene was produced, accounted for 61.2 mg/L (lye) and 64.1 mg/L (washing waters) (82–88% of total carotenoid content). Above all, enriched streams had a noteworthy stimulating effect on the β-carotene synthesis, because both the maximum β-carotene yield per volume of enriched effluents and specific β-carotene production rate were higher when compared with the respective values obtained from trials with synthetic reference medium without added effluents. Hydroxytyrosol and tyrosol showed high stability during the non-sterile process for β-carotene production by B. trispora grown in the enriched effluents. This finding strengthens the potential toward the generation of multiple high-value products, which could lower the natural β-carotene production costs.

The effect of ultrasound treatment on the extraction of lycopene and β-carotene from cherry silverberry fruits

The aim of this study was to evaluate the effect of ultrasound treatment on the efficiency of carotenoid extraction from cherry silverberries. Fruits (except for the control group) were sonicated at three power levels: 200,400 and 600 W. Fruits were extracted in 95% ethyl alcohol. After extraction, the content of β-carotene and lycopene in the extract was determined by HPLC with the UV-Vis SPD-20A detector and an acetonitrile-methanol-dichloromethane (75:25:5) as mobile phase. The dry matter content of all three sonicated samples was lower than in the control sample. Sonication at 200 W significantly (p < 0.05) increased β-carotene yield (by 59%) relative to the control sample. Lycopene yield also increased significantly (p < 0.05) relative to the control group after sonication at 200 W, but further increase in power did not induce significant improvement in extraction efficiency. The results of this study indicate that ultrasound treatment at 200 W (0.25 W·cm−3) increases the efficiency of carotenoid extraction from cherry silverberries and higher power can lead to reduction of extraction efficiency.

Effects of different light regimes on Dunaliella salina growth and β-carotene accumulation

Outdoor cultivations of microalgae for commercial scale production usually rely on natural sunlight following ‘Sine’ regime, but most lab scale studies adopt ‘Block’ or ‘Continuous’ regimes, leading to poor prediction of outdoor cultivation results. To fill this gap on β-carotene production by Dunaliella salina, the effects of light regimes (‘Block’, ‘Sine’, ‘Continuous’) on cell growth and β-carotene accumulation in D. salina were investigated in this study. The results showed significant difference between these regimes. When receiving the same amount of illumination, ‘Sine’ mode inferred 10–55% higher biomass yield and 31–58% higher β-carotene yield than those under the ‘Block’ mode. The biomass concentration, as well as protein and total fatty acid (TFA) content, has no significant difference between different light regimes, but the starch content in the ‘Continuous’ light regime was higher than that of ‘Sine’ and ‘Block’ regime. β-carotene accumulation under ‘Continuous in summer’ and ‘Simulated diurnal irradiance in summer’ regimes were correlated with increase (C18:1, C18:2) and decrease (C16:4, C18:3) of specific fatty acids (FAs) contents, rather than TFA content. Meanwhile, it was found that β-carotene accumulation was correlated with periodical Fv/Fm fluctuations under different light regimes, and indicate that exposure to periodical strong light is more favorable for β-carotene accumulation, rather than continuous light. These results indicated the significance of using ‘Sine’ light regime since it not only provided better predictions on outdoor production, but also helpful for increase biomass and β-carotene productivities.

Microalgae based biorefinery: Assessment of wild fresh water microalgal isolate for simultaneous biodiesel and β-carotene production

Microalgal biorefineries that produce biofuels and value-added products have been extensively investigated in recent years. This study has assessed potential of freshwater microalga for lipids and β-carotene production. The phylogenetic positioning of isolated strain by 18S rRNA sequence analysis revealed that it belonged to genus Scenedesmus. The strain was named Tetradesmus obliquus SGM19. Optimum growth conditions (nitrogen source, NaNO3 = 1.5 g/L; photoperiod 12L:12D) yielded biomass concentration = 2.5 g/L, β-carotene yield = 0.67 mg/g dry biomass, lipid content = 28.5 wt%. Antioxidant activity of β-carotene (measured using DPPH assay) was 69%. ~79% constituents of lipids could be identified from GC–MS analysis of biodiesel synthesized from the lipids. Major fatty acids present in the lipids were palmitic, heptadecanoic, linoleic, linolenic and arachidic acids. Properties of biodiesel estimated from FAME composition (e.g. cetane number = 48.59) are within limits of ASTM D6751 and EN 14214 standards.

β-Carotene Production from Dunaliella salina Cultivated with Bicarbonate as Carbon Source.

Bicarbonate has been considered as a better approach for supplying CO2 to microalgae cells microenvironments than gas bubbling owing t°Cost-effectiveness and easy operation. However, the β-carotene production was too low in Dunaliella salina cultivated with bicarbonate in previous studies. Also, the difference in photosynthetic efficiency between these tw°Carbon sources (bicarbonate and CO2) has seldom been discussed. In this study, the culture conditions, including NaHCO3, Ca2+, Mg2+ and microelement concentrations, were optimized when bicarbonate was used as carbon source. Under optimized condition, a maximum biomass concentration of 0.71 g/l and corresponding β-carotene content of 4.76% were obtained, with β-carotene yield of 32.0 mg/l, much higher than previous studies with NaHCO3. Finally, these optimized conditions with bicarbonate were compared with CO2 bubbling by online monitoring. There was a notable difference in Fv/Fm value between cultivations with bicarbonate and CO2, but there was no difference in the Fv/Fm periodic changing patterns. This indicates that the high concentration of NaHCO3 used in this study served as a stress factor for β-carotene accumulation, although high productivity of biomass was still obtained.

Methods for and pigments extration and determination of color in tomato for processing cultivars

Abstract The aim of this research was compare methods for pigments extraction and color determination in different tomato for processing cultivars, and choose the most promising materials for the industry. The tomatoes for processing studied were of the IT761, H9992, H9553, AP533, Advance, N901, BRSena, U2006, HY26, HY37 and HY68 cultivars. The extraction yields of lycopene and β-carotene using acetone and the mixture of ethanol, acetone and hexane (10:10:20) were determined, and the parameters of color were determined by colorimetric and digitals image methods (Cielab and Hunterlab), besides other physicochemical characteristics. The mixture of solvents is more efficient in the extraction of lycopene and β-carotene than acetone on its own. The methods of assessing the color through image presented higher levels of luminosity and chrome a* and b* than the instrumental method, given that the Hunterlab is closer to the colorimeter method, than Cielab. The analysis through digital images indicates that the procedure is promising, and it can be used as a instrument for analysing tomato colors. Considering that the level of TSS is fundamental to industrial performance, the IT761, BRSena, AP543, and H9992 cultivars stand out, moreover these cultivars have higher levels of lycopene and darker red color.

CAROTENE YIELD IN SWEET POTATO AFTER POTASSIUM AND PHOSPHORUS FERTILISER APPLICATION

ABSTRACT Sweet potato is one of the most important foods in the world. Among the different cultivated genotypes, it is worth highlighting those with orange pulp due to their ability to supply adequate amounts of provitamin A carotenoids to humans. This study evaluates the effect of potassium and phosphorus fertiliser application on carotenoid yield in sweet potatoes. Two experiments were carried out in São Luis City, Maranhão State, Brazil. The experimental design was a randomised block design, with six treatments for each of the studies (0, 30, 60, 90, 120 and 150 kg ha-1 K2O and 0, 60, 120, 180, 240 and 300 kg ha-1 P2O5), with four replicates. Cultivar 'Beauregard' was used, which has high levels of carotenoids. Roots were harvested at 123 days after planting for both experiments. The following variables were evaluated: root fresh and dry weight, dry weight content, in addition to the yield and contents of total carotenoids, α-carotene, β-carotene, 13-cis β-carotene and 9-cis β-carotene. With the average data of the evaluated characteristics, we performed analysis of variance and polynomial regression analysis. Potassium rates influenced carotenoid yield in sweet potato roots, with the rates of 73 to 77 kg ha-1 K2O being recommended to maximise the production of total carotenoids, β-carotene, and α-carotene. Phosphorus, in turn, influenced only total carotenoids, and is recommended at the rate of 174.09 kg ha-1 P2O5 to maximise the production of these nutrients.

Investigations in ultrasonic enhancement of β-carotene production by isolated microalgal strain Tetradesmus obliquus SGM19.

Microalgae constitute relatively novel source of lipids for biodiesel production. The economy of this process can be enhanced by the recovery of β-carotenes present in the microalgal cells. The present study has addressed matter of enhancement of lipids and β-carotene production by microalgal species of Tetradesmus obliquus SGM19 with the application of sonication. As first step, the growth cycle of Tetradesmus obliquus SGM19 was optimized using statistical experimental design. Optimum parameters influencing microalgal growth were: Sodium nitrate = 1.5 g/L, ethylene diamine tetraacetic acid = 0.001 g/L, temperature = 28.5 °C, pH = 7.5, light intensity = 5120 lux, β-carotene yield = 0.67 mg/g DCW. Application of 33 kHz and 1.4 bar ultrasound at 10% duty cycle was revealed to enhance the lipid and β-carotene yields by 34.5% and 31.5%, respectively. Kinetic analysis of substrate and product profiles in control and test experiments revealed both lipid and β-carotene to be growth-associated products. The intracellular NAD(H) content during late log phase was monitored in control and test experiments as a measure of relative kinetics of intracellular metabolism. Consistently higher NAD(H) concentrations were observed for test experiments; indicating faster metabolism. Finally, the viability of ultrasound-exposed microalgal cells (assessed with flow cytometry) was >80%.

Antioxidant composite films with chitosan and carotenoid extract from Chlorella vulgaris: optimization of ultrasonic-assisted extraction of carotenoids and surface characterization of chitosan films

Recently, there is an increasing interest towards the application of bioactive natural polymer based packaging materials instead of synthetic polymers. Therefore, the aim of this work was to investigate to edible, biodegradable and antioxidant chitosan film incorporated with carotenoid extracts from Chlorella vulgaris. Firstly, green extraction technology, ultrasonic-assisted extraction (UAE) was applied for the extraction of lutein, β-carotene, and α-carotene from Chlorella vulgaris. Response surface methodology (RSM) was used for optimizing the extraction parameters of α-carotene, β-carotene and lutein. The maximum yields of lutein, β-carotene, and α-carotene were 4.844 ± 0.780, 0.258 ± 0.020 and 0.275 ± 0.040 mg g−1, respectively. Secondly, the produced chitosan films were characterized by FT-IR and SEM techniques. And also the release study of chitosan films was investigated according to the total phenolic content and antioxidant capacity methods. Moreover, the addition of carotenoid extract significantly improved biodegradable films that can be used as a packaging material and patches because of its antioxidative properties.

Optimization of bioactive compounds extraction assisted by microwave parameters from Kappaphycus alvarezii using RSM and ANFIS modeling

The main focus of present study is to optimize the extraction parameters for maximum yield of biomass, β-carotene and chlorophyll content and their antioxidants from Kappaphycus alvarezii by response surface methodology (RSM) along with adaptive neuro-fuzzy inference system (ANFIS). Central composite rotatable design of RSM was applied to optimize the effect of microwave-assisted extraction (MAE) parameters. In this way, four parameters, such as methanol concentration (X1: 65–80%), microwave power (X2: 15–20%), radiation induced temperature (X3: 45–60 °C) and radiation exposure time (X4: 7.5–15 min) at five levels were selected. Optimal conditions were found as X1 = 80%, X2 = 20%, X3 = 45 °C and X4 = 12.5–14.5 min, under this condition, % yield of biomass (y1) = 86.231–87.212%, β-carotene and chlorophyll content (y2) = 77.348–82.841 mg/gm, %DPPHsc (y3) = 69.362–72.768%, %ABTSsc (y4) = 66.282–70.621%, FRAP (y5) = 62.654–64.381 μg mol (Fe(II))/g has been obtained. Also, optimized values of MAE parameters were compared and verified with ANFIS. Further, the major bioactive compounds present in the optimally obtained extract of K. alvarezii was identified through FTIR, gas chromatography-mass spectrum and HPLC.

Two-step strategy for obtaining dunaliella sp. biomass and β-carotene from anaerobically digested poultry litter wastewater

Using anaerobically digested wastewater to culture microalgae can realize wastewater treatment and biomass acquisition simultaneously. However, previous works concentrated mostly on wastewater treatment or bioenergy production, whereas few studies carried out high value-added product production. Therefore, this study focused on using anaerobically digested poultry litter wastewater (PLW) for obtaining biomass and β-carotene. The feasibility and optimal conditions for pollutant removal, biomass production, and β-carotene accumulation were investigated. The results showed that Dunaliella FACHB-558 could utilize ammonium as the sole nitrogen source. With the optimal conditions of 36 psμ salinity and 200 μmol/m2/s light intensity, the total nitrogen, total phosphorus, and total organic carbon removal, and biomass and β-carotene yields reached 63.8%, 87.2%, 64.1%, 678 mg/L, and 4.02 mg/L, respectively, after 15 days of treatment. To further improve β-carotene accumulation, a two-step strategy was adopted. By changing the culture medium from the anaerobically digested PLW to modified BG-11 medium and applying optimal culture conditions of 180 psμ salinity, 14 mg/L nitrogen, and 250 μmol/m2/s light intensity in the second step, the β-carotene yield reached 7.26 mg/L, 42.4% more than in the first step. Moreover, the salinity was found to be the most crucial factor influencing β-carotene accumulation in the second step.

Optimization of submerged fermentation process for improved production of β-carotene by Exiguobacterium acetylicum S01

Carotenoids are natural pigments with substantial applications in nutraceutical, pharmaceutical, and food industries. In this study, optimization of the fermentation process for enhancement of β-carotene and biomass production by Exiguobacterium acetylicum S01 was achieved by employing statistical designs including the Placket-Burman design (PBD) and response surface methodology (RSM). Among the seven variables investigated by two levels in PBD, glucose, peptone, pH and temperature were indicated as crucial variables (p < 0.0001) for β-carotene and biomass productivity. Response surface methodology was further applied to evaluate the optimal concentrations of these four variables for maximum β-carotene and biomass productivity. The optimized medium contained glucose 1.4 g/L, peptone 26.5 g/L, pH 8.5, and temperature 30 °C, respectively. A significant increase in β-carotene (40.32 ± 2.55 mg/L) and biomass (2.19 ± 0.10 g/L) productivities in E. acetylicum S01 were achieved by using RSM, which was 3.47-fold and 2.36-fold higher in the optimized medium compared to the un-optimized medium. Further, the optimum fermentation condition in the 5-L bioreactor was achieved a maximal β-carotene yield of 107.22 ± 5.78 mg/L within 96 h. Moreover, the expression levels of carotenoid biosynthetic genes (phytoene desaturase (CrtI) and phytoene synthase (CrtB)) were up-regulated (2.89-fold and 3.71-fold) in E. acetylicum under the optimized medium conditions. Overall, these results suggest that E. acetylicum S01 can be used as a promising microorganism for the commercial production of β-carotene.

Process-intensified extraction of phycocyanin followed by β-carotene from Spirulina platensis using ultrasound-assisted extraction

ABSTRACTThe development of sustainable technology for the extraction of value-added products from the ecological feedstock is gaining importance. The present work focuses on the intensification of extraction of different metabolites such as phycocyanin and β carotene from microalgae (Spirulina platensis) and also presents the optimum extraction parameters using an ultrasound-assisted approach. Initially, phycocyanin was extracted from lyophilized biomass of Spirulina platensis using an ultrasound-assisted approach, and then the extract was centrifuged and the supernatant was treated by using the sugaring-out method to separate phycocyanin from chlorophyll. The remaining biomass was further used for the extraction of β-carotene using the same ultrasound-assisted approach. Various operating parameters such as amplitude, duty cycle, sonication time, and depth of horn immersed into the solution have been investigated for intensified extraction. Moreover, Taguchi design of experiments was used to evaluate the yield of β-carotene using the L9 orthogonal array based on the effect of probe length, amplitude, duty cycle, and sonication time. On the basis of results and signal-to-noise (S/N) ratio analysis, a ranking of the parameters was performed. It can be noted here that the order of ranking was amplitude > sonication time > duty cycle > depth of horn immersing into the solution. Overall, it has been observed that maximum extraction of phycocyanin and β-carotene was 67 mg/g of dry biomass and 4.66 mg/g of dry weight, which was obtained at optimal amplitudes of 80% and 66% of the duty cycle and 0.5 cm depth of horn immersing into the solution in 4 min and 10 min, respectively. The present study clearly demonstrated that significant intensification benefits can be obtained in terms of the extraction of metabolites at optimal conditions using the ultrasound-assisted approach.

Enhancement of docosahexaenoic acid (DHA) and beta-carotene production in Schizochytrium sp. using symbiotic relationship with Rhodotorula glutinis

The aim of the study is to enhance the docosahexaenoic acid (DHA) and beta-carotene production yields in Schizochytrium sp. by forming a symbiotic relationship with yeast cell Rhodotorula glutinis. Schizochytrium sp. is one of the most studied microalgae for DHA production. On the other hand, Rhodotorula glutinis is well known as carotenoid-producing yeast. Here, mixed culture of Schizochytrium sp. and Rhodotorula glutinis was used, for the first time, to enhance the yields for DHA and beta-carotene. The cells were recovered, biomass, lipid content, DHA and beta-carotene yields were analyzed. GC-FID and HPLC were used for lipid content analysis and beta-carotene yield, respectively. Cell dry weight was increased ∼2.6 fold while DHA and β-carotene yields were increased ∼1.18 and ∼1.76 fold respectively in co-culture medium compared to Schizochytrium-monoculture. The results highlight the possibility of using heterotroph microalgae and yeast cells in co-culture environment to enhance DHA yield along with β-carotene production.

Construction of high-yield strain by optimizing lycopene cyclase for β-carotene production

To optimize key enzymes, such as to explore the gene resources and to modify the expression level, can maximize metabolic pathways of target products. β-carotene is a terpenoid compound with important application value. Lycopene cyclase (CrtY) is the key enzyme in β-carotene biosynthesis pathway, catalyzing flavin adenine dinucleotide (FAD)-dependent cyclization reaction and β-carotene synthesis from lycopene precursor. We optimized lycopene cyclase (CrtY) to improve the synthesis of β-carotene and determined the effect of CrtY expression on metabolic pathways. Frist, we developed a β-carotene synthesis module by coexpressing the lycopene β-cyclase gene crtY with crtEBI module in Escherichia coli. Then we simultaneously optimized the ribosome-binding site (RBS) intensity and the species of crtY using oligo-linker mediated DNA assembly method (OLMA). Five strains with high β-carotene production capacity were screened out from the OLMA library. The β-carotene yields of these strains were up to 15.79-18.90 mg/g DCW (Dry cell weight), 65% higher than that of the original strain at shake flask level. The optimal strain CP12 was further identified and evaluated for β-carotene production at 5 L fermentation level. After process optimization, the final β-carotene yield could reach to 1.9 g/L. The results of RBS strength and metabolic intermediate analysis indicated that an appropriate expression level of CrtY could be beneficial for the function of the β-carotene synthesis module. The results of this study provide important insight into the optimization of β-carotene synthesis pathway in metabolic engineering.

Discovery of Several Novel Targets that Enhance β-Carotene Production in Saccharomyces cerevisiae.

β-Carotene is the precursor of vitamin A, and also exhibits multiple pharmaceutical functions by itself. In comparison to chemical synthesis, the production of β-carotene in microbes by metabolic engineering strategy is relatively inexpensive. Identifying genes enhancing β-carotene production in microbes is important for engineering a strain of producing higher yields of β-carotene. Most of previous efforts in identifying the gene targets have focused on the isoprenoid pathway where the β-carotene biosynthesis belongs. However, due to the complex interactions between metabolic fluxes, seemingly irrelevant genes that are outside the isoprenoid pathway might also affect β-carotene biosynthesis. To this end, here we provided an example that several novel gene targets, which are outside the isoprenoid pathway, have improving effects on β-carotene synthesis in yeast cells, when they were over-expressed. Among these targets, the class E protein of the vacuolar protein-sorting pathway (Did2) led to the highest improvement in β-carotene yields, which was 2.1-fold to that of the corresponding control. This improvement was further explained by the observation that the overexpression of the DID2 gene generally boosted the transcriptions of β-carotene pathway genes. The mechanism by which the other targets improve the production of β-carotene is discussed.

Progress in metabolic engineering of β-carotene synthesis

β-carotene is an important natural plant pigment and has various physiological functions in organisms. With the proposition of systematic biology and progress in carotenoids biosynthesis since the 1960s, metabolic engineering has played a significant role in enhancing carotenoid production. In this review, we present β-carotene's traditional production methods and metabolic engineering strategies for constructing β-carotene-producing strains. Meanwhile, main problems and corresponding solutions to improve β-carotene yield of engineered strains were further analyzed, for further efficient microbial production of β-carotene.

Evaluation of microalgae production coupled with wastewater treatment

ABSTRACTIn the present study, the feasibility of microalgae production coupled with wastewater treatment was assessed. Continuous cultivation of Chlorella sorokiniana with wastewater was tested in lab-scale flat-panel photobioreactors. Nitrogen and phosphorus removals were found to be inversely proportional to the four dilution rates, while chemical oxygen demand removal was found to be 50% at all the tested conditions. The biomass obtained at the highest dilution rate was characterized for its content of lipids, proteins and pigments. The average yields of fatty acid methyl esters (FAMEs), protein, lutein, chlorophylls and β-carotene was 62.4, 388.2, 1.03, 11.82 and 0.44 mg per gram dry biomass, respectively. Economic analysis revealed that potentially more than 70% of revenue was from the production of pigments, that is, chlorophyllin (59.6%), lutein (8.9%) and β-carotene (5.0%) while reduction in discharging costs of the treated wastewaters could account for 19.6% of the revenue. Due to the low market price of biodiesel, the revenue from the above was found to be the least profitable (1.4%). Even when combining all these different revenues, this cultivation strategy was found with the current prices to be uneconomical. Power consumption for artificial light was responsible for the 94.5% of the production costs.