Yield Of Limonene Research Articles

Systems biology approach for enhancing limonene yield by re-engineering Escherichia coli

Engineered microorganisms have emerged as viable alternatives for limonene production. However, issues such as low enzyme abundance or activities, and regulatory feedback/forward inhibition may reduce yields. To understand the underlying metabolism, we adopted a systems biology approach for an engineered limonene-producing Escherichia coli strain K-12 MG1655. Firstly, we generated time-series metabolomics data and, secondly, developed a dynamic model based on enzyme dynamics to track the native metabolic networks and the engineered mevalonate pathway. After several iterations of model fitting with experimental profiles, which also included 13C-tracer studies, we performed in silico knockouts (KOs) of all enzymes to identify bottleneck(s) for optimal limonene yields. The simulations indicated that ALDH/ADH (aldehyde dehydrogenase/alcohol dehydrogenase) and LDH (lactate dehydrogenase) suppression, and HK (hexokinase) enhancement would increase limonene yields. Experimental confirmation was achieved, where ALDH-ADH and LDH KOs, and HK overexpression improved limonene yield by 8- to 11-fold. Our systems biology approach can guide microbial strain re-engineering for optimal target production.

Identifying Key In Silico Knockout for Enhancement of Limonene Yield Through Dynamic Metabolic Modelling.

Living cells display dynamic and complex behaviors. To understand their response and to infer novel insights not possible with traditional reductionist approaches, over the last few decades various computational modelling methodologies have been developed. In this chapter, we focus on modelling the dynamic metabolic response, using linear and nonlinear ordinary differential equations, of an engineered Escherichia coli MG1655 strain with plasmid pJBEI-6409 that produces limonene. We show the systems biology steps involved from collecting time-series data of living cells, to dynamic model creation and fitting the model with experimental responses using COPASI software.

Machine learning-driven prediction and optimization of pyrolysis oil and limonene production from waste tires

The pyrolysis oil from waste tires possesses significant economic value and is a crucial factor in determining the industrial viability of tire pyrolysis processes. Limonene in pyrolysis oil is a significant component with extremely high industrial application value. In the process of tire pyrolysis, predicting the pyrolysis products through operating conditions and feedstock composition can effectively control industrial operations and enhance operational efficiency. However, there is currently a lack of robust prediction methods for pyrolysis oil and limonene yield. This study proposes the application of machine learning to predict the yield of tire pyrolysis oil and limonene. Artificial Neural Network (ANN) and Random Forest (RF) models were developed to create prediction models. In the statistical analysis, RF achieved optimal R2 median values of 0.83 and 0.64 for pyrolysis oil and limonene predictions during the testing stage.For the prediction of limonene yield, the best R2 and RMSE values in the testing and training stages were 0.844, 3.76, and 0.964, 1.91, respectively. For the prediction of pyrolysis oil yield, the corresponding values were 0.926, 4.1, and 0.985, 1.889, in the testing and training stages, respectively.Temperature was identified as the most critical operating condition affecting pyrolysis oil and limonene yields, with relative importance percentages of 20.6% and 12.2% for oil and limonene yields, respectively. The optimal operating parameter range conducive to limonene yield is as follows: temperature between 350 and 450 °C, residence time between 0 and 50 min, and tire particle size greater than 15 mm.This study lays the foundation for the production of oil and the extraction of limonene from tire pyrolysis.

Production of limonene and its derivative in Saccharomyces cerevisiae via metabolic engineering

Limonene and its derivative perillic acid are widely used in food, cosmetics, health products, medicine and other industries as important bioactive natural products. However, inefficient plant extraction and high energy-consuming chemical synthesis hamper the industrial production of limonene and perillic acid. In this study, limonene synthase from Mentha spicata was expressed in Saccharomyces cerevisiae by peroxisome compartmentalization, and the yield of limonene was 0.038 mg/L. The genes involved in limonene synthesis, ERG10, ERG13, tHMGR, ERG12, ERG8, IDI1, MVD1, ERG20ww and tLS, were step-wise expressed via modular engineering to study their effects on limonene yield. The yield of limonene increased to 1.14 mg/L by increasing the precursor module. Using the plasmid with high copy number to express the above key genes, the yield of limonene significantly increased up to 86.74 mg/L, which was 4 337 times higher than that of the original strain. Using the limonene-producing strain as the starting strain, the production of perillic acid was successfully achieved by expressing cytochrome P450 enzyme gene from Salvia miltiorrhiza, and the yield reached 4.42 mg/L. The results may facilitate the construction of cell factory with high yield of monoterpene products by S. cerevisiae.

Sphaerotilus natans hemoglobins have an NADH oxidation activity and promote the yield of limonene in an engineered E. coli strain

Bacterial hemoglobins play important roles inside the cell. Phylogenetically, they belong to three different families: the single domain hemoglobin, flavohemoglobin and truncated hemoglobin. Vitreoscilla hemoglobin (VHb) is the first characterized bacterial hemoglobin, and belongs to the single domain hemoglobin family. Heterologous expression of VHb promotes the growth of host cells under microaerobic conditions, and enhances the yield of products during fermentation. Although VHb has been widely applied in the biotechnology field, other bacterial hemoglobins have not demonstrated similar applications. In this study, we identified four bacterial hemoglobins from the microaerobic growing bacterium Sphaerotilus natans, including one flavohemoglobins (FHB) and three truncated hemoglobins (THB1, THB2 and THB3). Absorption spectrum studies validate the existent of the Soret peak and Q-band characteristic to heme and suggest heme groups in FHB and THB1 are hexa- or penta-coordinated, respectively. Our studies demonstrate that FHB and all three truncated hemoglobins have NADH oxidation and radical production activities, which is surprising since truncated hemoglobins do not have a reductase domain that could bind NADH. However, the M. tuberculosis HbN does not show these activities, indicating they are not universal among truncated hemoglobins. Docking studies suggest the nicotinamide ring of NADH may bind to the distal heme pocket of THB1, suggesting the direct electron transfer from NADH to heme might be possible. Our truncated hemoglobins also show peroxidase activities that in THB2 and THB3 could be inhibited by FdR, indicating possible interactions between FdR and truncate hemoglobins. Expression of FHB and THB1 in E. coli could promote cell growth. THB1 also enhances the production of limonene in an engineered E. coli strain, while VHb does not have this effect, which suggests that studies on truncated hemoglobins may lead to the discovery of new and more powerful tools that could have profound impact on biotechnology.

Advances in understanding kinetic mechanisms underlying waste ground tyre rubber pyrolysis

The depletion of natural resources and the need to reduce solid waste in urban areas have necessitated the incorporation of used materials such as waste ground tyre rubbers (WGTR), into manufacturing processes. As a result, techniques and recycling methods have been established to use tyres as feedstock for marketable products since tyres have a calorific value higher than coal and contain a significant amount of carbon black. Among several techniques, pyrolysis has emerged as the most appealing for treating WGTRs. This technique allows the recovery of valuable products like combustible gases, fuels and chemicals, and activated carbon. Studies have focused on understanding the mechanism underlying the WGTR pyrolysis through the establishment of mathematical models and reaction patterns to valorise WGTRs and efficiently produce marketable chemicals. This paper presents an overview of recent developments in understanding WGTR pyrolysis mechanisms. A general mechanism observed involves a first depolymerisation/condensation of the rubbers, then a degradation of the condensed products, and finally a devolatilisation of additives. Based on the limited information available on the chemicals' formation mechanism, it is assumed that limonene and isoprene are derived from natural rubber (NR), through a series of β-scission and depropagation reactions of polyisoprene and intramolecular cyclisation and scission of monomeric isoprene, respectively, with an equilibrium step of Diels-Alder reaction. The maximum yield of limonene and isoprene have been found to be 51% and 20.5% at temperature around 500°C respectively. The isoprene yield can be increased up to 37.57 % with the use of catalyst such as Calcium Oxide.

Conventional vs. Microwave-Assisted Hydrodistillation: Influence on the Chemistry of Sea Fennel Essential Oil and Its By-Products.

The main objectives of this study were to investigate the effects of the applied essential oil (EO) isolation method, conventional hydro-distillation (HD), and microwave-assisted hydro-distillation (MHD) on the chemical profile of sea fennel (Crithmum maritimum L.) essential oil and to investigate the main constituents present in the liquid by-products of EOs isolation (hydrolate and residual wastewater). Headspace-solid phase microextraction (HS-SPME) was used to isolate hydrolate components, while gas chromatography coupled with mass spectrometry (GC-MS) was used to detect and analyse the chemical constituents of the essential oils and hydrolates. The phenolic composition of the wastewater extracts was analysed by high performance liquid chromatography (HPLC). The EO obtained by MHD had a higher yield of limonene and sabinene. The chemical composition of the hydrolates differed from the EO compositions. The content of terpinen-4-ol in the MHD hydrolate was higher, while several compounds were detected in relatively high proportions only in the HD hydrolate. MHD also resulted in a higher phenolic content of the wastewater, where an increase in the concentration of chlorogenic acid was also observed. It can be concluded that the isolation method had a great influence on the profile of sea fennel EOs, especially on their corresponding hydrolates and residual wastewater extracts. Due to their valuable chemical composition, these by-products can be a cost-effective source of bioactive compounds that have great potential for use in various industries.

Consecutive recovery of recovered carbon black and limonene from waste tyres by thermal pyrolysis in a rotary kiln

End of life tyres are one of the most abundant and least recycled waste streams. Material recovery processes that accommodate for the complexity and heterogeneity of waste tyres, such as pyrolysis, require urgent development. Examples of recycled tyre pyrolysis products include recovered carbon black (RCb), a recycled filler, and limonene, a solvent, although investigations reporting the recovery of both are limited. In this study the pyrolysis of waste tyres was investigated in a batch rotary kiln under varying conditions (temperature, gas flow, sample mass). Characterisation analysis was then applied to selected samples of RCb (BET surface area, transmissibility of toluene extract, proximate analysis) and tyre pyrolysis oil (yield, limonene content, FTIR). The best RCb (77.7 m2/g, 0.2% volatile matter, 17.0% ash, 94.8% transmissibility of toluene extract) was produced at 550 °C with a high ratio of purge gas flow (2000 mL/min) to rubber sample mass pyrolysed (100 g), minimising the recondensation of tarry volatiles on the RCb. The highest oil and limonene yield (38.4% and 17.4 g/kg waste tyres respectively) was produced at a temperature of 550 °C with a low ratio of purge gas (400 mL/min) to rubber sample pyrolysed (200 g), as these conditions prevented the decomposition of limonene to less valuable products and improved the oil trap condensation efficiency. These findings demonstrate the importance of pyrolysis processing conditions when considering the consecutive production of RCb and solvents from waste tyre pyrolysis, which shows significant future potential to valorise waste tyres, incentivising recycling.

Effective synthesis of limonene over mild alkali-modified 13X catalysts in α-pinene isomerization

The mild alkali-treated 13X was employed in the liquid phase isomerization reaction of α-pinene to increase the yield of limonene. A maximum α-pinene conversion of 100% and limonene yield of 59.1% could be achieved. Crystallinity could be well retained, and the amounts of medium-strong acid sites and the distribution of Brønsted and Lewis acid sites could be changed after the mild alkali treatment for 13X zeolite. Samples with higher micropore specific surface area were easier to obtain higher yields of limonene due to shape-selectivity. A proper amount of the medium-strong acid and the synergistic effect of Brønsted and Lewis acid sites were beneficial to enhance the catalytic activity and the yield of monocyclic product limonene. The main reason for catalyst deactivation could be the decrease in the active sites due to soft coke deposition. For the regeneration process, the conversions of α-pinene were between 95% and 99% during the first seven cycles and the conversion dropped to 90.6% after the eighth cycle, which demonstrated that the ethanol flushing and calcination could be an effective regeneration method for the 13X catalyst used in the α-pinene isomerization reaction.

Converting polyisoprene rubbers into bio-jet fuels via a cascade hydropyrolysis and vapor-phase hydrogenation process

Producing alternative drop-in bio-jet fuels from biomass provides a promising approach to achieve carbon neutrality. To upcycle biomass-based polyisoprene rubbers into jet-fuel range C10 cycloalkane, we proposed a cascade hydropyrolysis and vapor-phase hydrogenation process in a flow-through two-stage pressurized fixed-bed reactor. The hydropyrolysis temperature in the first stage is of vital importance for the formation of primary limonene intermediate in the non-catalytic degradation of polyisoprene rubbers, and a reaction temperature of 460 °C maximized limonene yield to 588.6 mg/g for natural rubber and 546.2 mg/g for Eucommia rubber. Over a Pt/C catalyst loaded in the second stage, the limonene intermediate produced from the first-stage reactor can be completely hydrogenated, giving a 642.7 mg/g yield of jet-fuel range C10 cycloalkane with 83.6% selectivity. The depolymerization mechanism of polyisoprene rubbers was thoroughly studied, and a competitive reaction between limonene hydrogenation and limonene dehydrogenation was observed. This is the first report on producing C10 cycloalkane from natural rubbers via a cascade hydropyrolysis and hydrogenation process, providing a promising strategy to upcycle polyisoprene rubbers into bio-jet fuels.

Novel photothermal pyrolysis on waste tire to generate high-yield limonene

Waste tire is a typical solid municipal waste with large production capacity. Developing new low carbon-emission method to pyrolyze waste tire and produce high-value limonene is promising. Herein, a novel photothermal pyrolysis system was successfully built, and waste tire was firstly pyrolyzed by photothermal at different temperatures (400 °C, 500 °C, 600 °C, and 700 °C). The photothermal pyrolysis can assist in achieving high yield of limonene, of which the yield can high up to 8.98 wt% at 600 °C and the relative peak areas of Gas Chromatography-Mass Spectrometer (GC–MS) results were all about 30 A.%. cis-1,4-polyisoprene in waste tire partly decomposed into some free radicals and they further reacted to generate limonene by intramolecular cyclization, dimerization, and isomerization. Parts of limonene aromatized to 1-methyl-4-(1-methylethenyl)-benzene and converted to triolefins that formed diene rings, the diene rings further aromatized to xylene and toluene, which were partly combined to generate 1,6,7-trimethyl-naphtalene. The photothermal pyrolysis mechanisms of waste tire were revealed: At higher pyrolysis temperature, waste tire partly decomposed and others polymerized to form amorphous carbons. The small aromatic rings and alkyl-aryl CC bonds further polymerized to be large aromatic rings system. The pores in char further formed and limonene generation was strengthened, because the limonene formation/decomposition was mainly limited in the pores. Subsequently, some pores were blocked at 700 °C, the generation of limonene was thus inhibited.

Study on the microwave extraction process and product distribution of essential oils from citrus peel

Citrus peel wastes containing a series of very beneficial active compounds are produced in large quantities around the world every year, which are usually extracted by traditional methods, requiring high energy costs, long extraction times and additional reagents. Based on the unique heating characteristics of microwave heating, this paper uses microwave heating to extract the essential oils of citrus peel. Exploring the content change rule and the composition distribution situation of citrus peel product with different reaction time and microwave power, and comparing the microwave extraction and conventional extraction methods of citrus peel essential oils, infer the mechanism of microwave extraction. Considering the efficiency and energy consumption, optimum microwave power and time were selected as 450 W and 10 min. At microwave power of 450 W, 29.22% liquid product was obtained with 37.82% limonene yield in the liquid. The limonene yield per unit energy consumption in case of microwave heating was found to be 18.6 times that of traditional heating, and the extraction mechanism based on microwave heating effect and pumping effect was proposed.

The Effect of Temperature and Particle Size on the Pyrolysis Products of Waste Tires and the Formation Mechanism of Limonene

The rapid development of the automotive industry has led to the accumulation of a large number of waste tires that contain a lot of reusable energy. Macromolecular organics in waste tires can be crack small molecule organics via pyrolysis. In this experiment, thermogravimetry (TG) and pyrolizer-gas chromatography/mass spectrometry (PY-GC/MS) were used to study the pyrolysis behavior of waste tires with different particle sizes, and the effect of temperature and particle size on the pyrolysis products of waste tires under low-temperature pyrolysis conditions, respectively. The volatile substances in waste tires decomposed intensively at 300-500�C and were completely pyrolyzed at 500�C. The content of limonene in the pyrolysis product was significant, and the yield of limonene could reach 27.73% when the waste tire particles were 0.180-0.250 mm and the pyrolysis temperature was 380�C. The mechanism of limonene formation from waste tires was discussed. This study indicated that raw material particle sizes and pyrolysis temperature could change the components and content of pyrolysis products.

Extracción enzimática de limoneno, limonina y otros compuestos relevantes a partir de subproductos de Citrus sinensis (naranja) y Citrus aurantiifolia (limón)

The byproducts from orange (Citrus sinensis) and lime (Citrus aurantiifolia) constitute nearly 50 wt. % of the fresh fruit and, unfortunately, these are discarded. These residues could be utilized to obtain extractable compounds of high added value, such as limonene and limonin. The extraction of limonene from peels and limonin from seeds, from both orange and lime, was evaluated with a commercial enzyme (Macerex). It was found that the yield of limonene was 17-fold higher (4.0 and 4.7 mg/g-peel from orange and lime respectively) using the enzymatic treatment as compared to blank. More bioactive compounds and in a higher concentration were obtained from peels using the enzymatic treatment as compared to blank. Limonin yield was twofold when the enzymes were applied to orange and lime seeds, as compared to blank (2.5 mg/g-orange seeds and 3.0 mg/g-lime seeds). The amounts of extracted limonene and limonin had a correlation with the amount of sugars released during degradation of the vegetal tissue. Enzymatic extraction of bioactive compounds from citrus by-products exhibits high yields, similar to traditional extraction treatment such as hydrodistillation, but under milder conditions.

A microfactory in yeast peroxisomes

Synthetic Biology Considerable synthetic biology efforts are focused on engineering yeast to produce valuable metabolites. Production in the cytosol can be challenging because of toxicity or crosstalk with cellular pathways. Dusseaux et al. harnessed the yeast peroxisome to produce geranyl diphosphate (GPP), a precursor to monoterpenoids, monoterpene indole alkoids, and cannabinoids. Targeting the entire pathway for GPP synthesis, along with an enzyme that converts GPP to the monoterpene limonene, to the peroxisome gives a 125-fold improvement in yield of limonene compared with production in the cytoplasm. Additional monoterpenes can be synthesized with the same platform using the appropriate monoterpene synthase. Peroxisomal production also facilitates downstream processing involving oxidation by cytochrome P450 enzymes. Peroxisome microfactories could be used for the modular assembly and optimization of other complex pathways. Proc. Natl. Acad. Sci. U.S.A. 117 , 31789 (2020).

Effects of pressure and residence time on limonene production in waste tires pyrolysis process

Pyrolysis of waste tires is a promising way to cost-efficiently produce high value limonene, but the reaction characteristics and mechanisms of limonene under pressurized pyrolysis are unclear to date. This study aims to investigate the effects of residence time and pressure on limonene conversion during waste tire pyrolysis. The yields of limonene increased significantly with a decrease of residence time during high pressure pyrolysis. At a pressure of 1.0 MPa, the yield of limonene increases by 20 % as the residence time decreases from 60 s to 15 s, while limonene concentration in TDO (tire-derived-oil) increases to 35.10 wt.%. On the other hand, the pressure has a positive effect on limonene production. The yield of limonene at 1.0 MPa is approximately 1.7 times larger than that of 0.1 MPa at a residence time of 30 s. Based on Spearman correlation analysis, the mechanisms of limonene conversion at pressurized pyrolysis were studied. Longer residence time means limonene undergoes more secondary reactions to be degraded to aromatic rings such as xylene and trimethylbenzene. However, the higher N2 partial pressure during pyrolysis suppressed the decomposition of limonene, producing TDO with high limonene concentration. Therefore, methods of reducing residence time and increasing pressures promote yield and concentration of limonene in the aspect of inhibiting decomposition, which provides references for improvement in limonene production by waste tire pyrolysis.

Waste Tyres Pyrolysis for Obtaining Limonene.

This review deals with the technologies of limonene production from waste tyre pyrolysis. Thermal decomposition is attractive for tackling the waste tyre disposal problem, as it enables both: energy to be recovered and limonene to be obtained. This material management recycling of tyres is environmentally more beneficial than the burning of all valuable products, including limonene. Given this recoverability of materials from waste tyres, a comprehensive evaluation was carried out to show the main effect of process conditions (heating rate, temperature, pressure, carrier gas flow rate, and type of volatile residence and process times) for different pyrolytic methods and types of apparatus on the yield of limonene. All the results cited are given in the context of the pyrolysis method and the type of reactor, as well as the experimental conditions in order to avoid contradictions between different researchers. It is shown that secondary and side reactions are very sensitive to interaction with the above-mentioned variables. The yields of all pyrolytic products are also given, as background for limonene, the main product reported in this study.

PRODUCTION AND ANALYSIS OF THE COMPOSITION OF SUPERCRITICAL CARBON DIOXIDE EXTRACTS FROM EXOCARPY CITRUS MEYERI TAN.

The purpose of this work is to evaluate the effectiveness of using supercritical carbon dioxide extraction to obtain essential oils from exocarpy of Meyer lemon in comparison with the pressing method.
 The plant material is represented by the exocarpy of Meyer lemon Citrus meyeri Tan., Rutovye family – Rutaceae, collected in the vicinity of Khujand (Republic of Tajikistan), in November 2014. Supercritical carbon dioxide extracts (CO2 extracts) were obtained at the Research Center for Ecological GORO resources (Rostov-on-Don) at the KOERS1 installation. As a comparison, we used the essential oil obtained by direct pressing on hydraulic presses without heating. Qualitative and quantitative analysis of the components was carried out by gas chromatography with chromatography-mass spectrometric detection.
 As a result of the study, it was found that the optimal parameters of CO2 extraction, which provide the highest content of limonene and γ-terpinene in the extract, are: 50 °С, 16 MPa, and 30 min. A comparative analysis of the content of extract components showed that the yield of limonene during supercritical extraction, compared with traditional technology, increased by 7.5%, the γ-terpinene content was 1.5 times. In addition, significant differences were found in the qualitative and quantitative composition of the essential oil and the CO2 extract of Meyer lemon. In essential oil, the content is higher: α- and β-pinene, p-cumene, geranial, and others. In the CO2 extract, the content is higher: sabinene, α-humulene, β-bisabolene and other low-volatile compounds, with an additional 9 components: α-thuen, terpenolene, geranyl acetate, etc., which indicates the need for an additional pharmacological study of the CO2-extract of Meyer lemon.

Surfactant-assisted enzymatic extraction of the flavor compounds from Zanthoxylum bungeanum

ABSTRACTSurfactant-assisted enzymatic hydrolysis, which has been rarely involved in food industry, was used to extract the flavor compounds from Zanthoxylum bungeanum. The extraction conditions optimized through orthogonal and single-factor experiments were as follows: the amount of cellulase, neutral protease and surfactant (calcium stearyl lactylate) addition was 0.5%, 0.3% and 0.7% (w/w), respectively, the solid–liquid ratio was 1:6 (g/mL), the enzymolysis temperature was 45°C, the enzymolysis time was 3 h and the granularity was less than 60 mesh. Under these conditions, the yield of limonene, linalool and alkylamide increased from 1.02% to 7.51%, from 16.50% to 48.39%, and from 27.40% to 37.74%, respectively. The mechanism of surfactant-assisted enzymatic hydrolysis to extract the flavor compounds were also investigated. The study provides a scientific basis for the refinement and comprehensive utilization of Zanthoxylum bungeanum.

Optimization of essential oil extraction from orange peels using steam explosion

In this work, a new extraction process using steam explosion at high temperature and pressure was developed, to drastically shorten the extraction time and improved extraction of the essential oil from citrus peels. In steam explosion process, the material is subjected to the high-pressure saturated steam following by substantially dropping the pressure through an angle valve to a vacuum tank. The optimum essential oil yield by the steam explosion was obtained at the 170 °C, 8 bar in 240 seconds duration time. The essential oil extraction of a certain amount of citrus peels by hydro-distillation took nearly eight times longer than explosion extraction process. The obtained citrus oil from hydro-distillation processes had 10 to 13 major components more than‏ the steam explosion, as shown by gas chromatography (GC-MS). The maximum product yield of Limonene, a major favorable component, were 77% and 100% in hydro-distillation and steam explosion processes, respectively.