Ethene (Ethylene)

Ethene, commonly known as ethylene, stands out as the leading organic chemical in production volume. This versatile compound serves as the foundational substrate for a myriad of products, ranging from various plastics to antifreeze solutions and different solvents.

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Figure 1  At the Grangemouth facility in Scotland, ethene is produced through the steam cracking of naphtha.
By kind permission of INEOS Manufacturing Scotland.

Key Applications of Ethene (Ethylene)

Ethene is primarily utilized for the production of:

• Polymers:
  • Poly(ethene)
  • Ethylbenzene, which leads to phenylethene and subsequently poly(phenylethene)
  • Chloroethene (vinyl chloride) resulting in poly(chloroethene), also known as poly(vinyl chloride) or PVC
  • Ethylbenzene to poly(phenylethene) resulting in polystyrene
• Other Chemical Compounds:
  • Epoxyethane leading to diols like ethane-1,2-diol
  • Ethanol

The main consumption of ethene is dedicated to polymer manufacturing. About 60% of the global ethene demand is attributed to poly(ethene) (HDPE 28%, LLDPE 18%, LDPE 14%), while dichloro-1,2-ethane serves as a precursor to chloroethene and therefore PVC, accounting for an additional 11%. Ethylbenzene, the pathway to poly(phenylethene), consumes another 5%1.

Approximately 16% of worldwide ethene production is directed towards generating epoxyethane (ethylene oxide).

Global Ethene (Ethylene) Production Statistics

Global production figures are as follows:

• World total: 134 million tonnes1
• Asia Pacific: 50 million tonnes1
• United States: 25 million tonnes2
• Europe: 20 million tonnes3
• Middle East: 20 million tonnes1

Data from:
1. Estimated from Nexant and ChemVision.
2. Guide to the Business of Chemistry, American Chemistry Council.
3. Petrochemicals Europe.

The Middle Eastern production largely originates from Saudi Arabia, contributing 17 million tonnes annually, with one particular refinery generating over 2 million tonnes yearly, making it the fourth largest ethene production facility globally.

Production Processes of Ethene (Ethylene)

Ethene is synthesized through the cracking of various fractions derived from the distillation processes of oil and natural gas. The common methods include:

• Steam cracking of ethane and propane sourced from natural gas and crude oil
• Steam cracking of naphtha extracted from crude oil
• Catalytic cracking of gas oil from crude oil

The choice of feedstock is influenced by availability, cost fluctuations, and the demand for other cracking by-products. The dominance of steam cracking is notable, with some facilities capable of producing 3,600 tonnes of ethene per day.

Figure 2  Ethene distribution through pipeline networks across Europe.

In Europe, surplus ethene is transported via pipelines that link to various chemical plants and refineries (Figure 2).

A new generation of crackers is being established in the United States to utilize the current excess supply of ethane and other hydrocarbon gases, a direct outcome of fracking. The U.S. produces approximately 25 million tonnes of ethene annually, with around 9 million tonnes sourced from ethane and another 4 million from propane. Projections indicate that by 2020, production from ethane could surge to about 14 million tonnes, while propane production might hit 5 million tonnes, influenced by fracking advancements.

Figure 3  The inaugural shipment of ethane from U.S. shale gas to Europe was relayed to the petrochemical site at Rafnes in Norway in March, followed by a notable shipment to Grangemouth, Scotland, a few months later. The first propane shipments are expected in 2016. Ethane, once stored at 283K, can serve as fuel but is also cracked to yield ethene and other alkenes. Utilizing propane as a feedstock typically yields a higher proportion of propene. Pictured is the Dragon, which stands as the world's largest gas tanker, with a capacity of 27,500 m3 of gas.

With kind permission of Ineos

In Brazil, new plants are emerging that utilize bioethanol sourced from sugar cane for ethene production. High yields of bio-based ethene are achieved by dehydrating ethanol vapors with a catalyst comprising a blend of magnesium oxide, alumina, and silica at temperatures ranging from 600-750 K:

The resulting ethene is primarily intended for the production of bio-based poly(ethene).

Date last updated: 4th January

The escalating international need for petroleum products, combined with its limited availability and adverse environmental impacts, has spurred the development of renewable fuels and chemicals compatible with existing infrastructure. Ethylene, a noteworthy potential renewable feedstock, is gaining attention. In 2019, the total global production capacity of ethylene was recorded at 142 million metric tonnes and is projected to rise to 165 million metric tonnes by 2025, potentially influencing the global economy by nearly US$200 billion annually. Ethylene is crucial in multiple sectors such as plastics, textiles, and solvents. Furthermore, it can be catalytically transformed into gasoline-like hydrocarbons within the C5-C10 range.

Current ethylene production predominantly involves steam cracking processes using fossil fuels or ethane dehydrogenation, making it the most significant CO2-emitting activity within the chemical industry. With each pound of ethylene produced, about 2 MJ of energy is consumed, signifying that this industry contributes approximately 1.5% of the carbon footprint in the United States. Transitioning to renewable ways of producing ethylene could thus meet substantial energy and chemical needs while promoting environmental sustainability.

Interestingly, ethylene can also be generated biologically. Functioning as a plant hormone, it influences plant growth and development while serving protective roles against abiotic and biotic stresses, including pest invasions. Within plants, ethylene is synthesized through a two-step reaction initiated by methionine, forming S-adenosyl-methionine (SAM). First, SAM is converted into 1-aminocyclopropane-1-carboxylic acid (ACC) by ACC synthase. Subsequently, ACC oxidase facilitates the release of ethylene and cyanide (CN). Although CN is converted to β-cyanoalanine to prevent toxicity within the plant, utilizing this pathway for ethylene production through other organisms is constrained by the necessity to manage the cyanide.

Additionally, diverse microorganisms, including certain bacteria and fungi, are known to produce ethylene, likely contributing to plant pathogenesis. In Escherichia coli, Cryptococcus albidus, and various other bacteria, ethylene can sporadically arise through the oxidation of 2-keto-4-methylthiobutyric acid (KMBA), a transaminated methionine derivative involved in a reaction enhanced during limited ammonia conditions (C/N=20). The production pathway of KMBA is assumed to reclaim amino nitrogen from methionine, leading to ethylene release from KMBA. Another production pathway occurs in Pseudomonas syringae and Penicillium digitatum, where α-ketoglutarate (AKG) and arginine serve as substrates in a reaction catalyzed by an ethylene-forming enzyme (EFE).

Expression of a singular efe gene sourced from P. syringae resulted in ethylene production in multiple hosts, including E. coli, Saccharomyces cerevisiae, Pseudomonas putida, Trichoderma viride, Trichoderma reesei, tobacco plants, and cyanobacteria. These organisms can utilize various carbon feedstocks, including lignocellulose as well as CO2, which underscores the potential diversity of substrates for bioderiving ethylene. Importantly, ethylene is non-toxic to these microorganisms, and due to its gaseous state, it can be easily separated from the cultures—an advantage over other biofuel products like alcohols or lipids that tend to be toxic or costly to extract.

Nonetheless, additional basic and applied research is vital to commercialize bioethylene production effectively. Emerging fields of study encompass an extensive analysis of EFE structure and reaction mechanisms, metabolic engineering approaches aimed at boosting productivity, and the design of ethylene harvesting technologies. This review seeks to collate existing literature while providing insights and strategies for future bioethylene research and development.

Discovery of EFE

Ethylene acts as a hormone, influencing numerous facets of plant growth and stress management. It is also a metabolic byproduct in multiple fungi and bacteria closely associated with plants. One of the earliest identified ethylene-producing microbes is the common green mold on citrus, P. digitatum. This was corroborated by preparing a cell-free system from P. digitatum, leading to the identification of EFE as a 42-kD protein that necessitates ferrous iron, oxygen, AKG, and arginine for ethylene synthesis. This mechanism contrasts with the two-enzyme approach employed by higher plants that utilize methionine as a precursor.

The initial reports of bacterial ethylene production came from strains of Pseudomonas solanacearum, which play roles in ripening bananas or causing wilting in tobacco and tomato plants. The most productive ethylene-producing microbes include specific pathovars of P. syringae, such as P. syringae pv. phaseolicola PK2 (Kudzu strain) and P. syringae pv. glycinea, both implicated in plant diseases affecting Kudzu and soybeans respectively. A cell-free system prepared from the Kudzu strain revealed that a 42-kD EFE monomer was essential for the reaction involving ferrous iron, oxygen, AKG, and arginine, aligning with EFE investigations in P. digitatum. Despite variations between N-terminal sequences of P. digitatum and Kudzu EFE, they share considerable sequence similarities. The Kudzu efe gene manifests on an indigenous plasmid, and its cloning and expression in E. coli confirmed that a single gene suffices for ethylene production in foreign hosts.

Diversity and Characteristics of EFE Sequences

To date, no comprehensive review on EFE sequence diversity exists despite substantial sequencing data availability. We thus constructed a phylogenetic tree of EFE using sequences from the NCBI database exhibiting over 40% similarity to that of Kudzu. These sequences are classified into two principal groups and one minor group; pairwise sequence comparisons reveal approximately 25% identity and 65% similarity. Notably, the central regions show the highest conservation (Wu Xu, unpublished data). The identified sequences were categorized as ethylene (succinate)-forming enzyme, 2-oxoglutarate (2OG)-Fe(II) oxygenase, hypothetical proteins, aminocyclopropane-1-carboxylic acid oxidase (ACCO), ACC deaminase, and oxidoreductase. This variety underscores the necessity for further functional investigations to categorize EFE and its related sequences accurately. Since both EFEs and ACCOs catalyze ethylene formation and belong to the superfamily of 2OG/Fe(II)-dependent hydroxylases, they may exhibit shared structural characteristics. To pinpoint conserved amino acids, we compared representative ACCO sequences from the Protein Data Bank (PDB) against Kudzu EFE. The leading two enzymes determined were an ACCO from petunia, the only ACCO with an experimentally resolved structure (PDB ID: 1W9Y; 29.7% similarity and 18.1% identity), and a putative 2OG-Fe(II) oxygenase from Caulobacter crescentus (PDB ID: 3OOX; 26.1% similarity and 17.1% identity). Other ACCOs from Arabidopsis thaliana and Zea mays, along with 2OG-Fe(II) oxygenases from the cyanobacteria Anabaena variabilis and Nostoc punctiforme, were also included for comparison. Notably, the cyanobacterial sequences are particularly intriguing as their 2OG-Fe(II) oxygenases might operate as EFE; these organisms serve as nitrogen-fixing symbionts with plants, and ethylene production could facilitate their symbiosis establishment. We identified 17 conserved amino acids shared among Kudzu EFE, ACCOs, and 2OG-Fe(II) oxygenases. These residues potentially play critical roles in the enzyme's structure and functionality. Research indicates that most active sites among 2OG/Fe(II)-dependent hydroxylase enzymes consist of a singular ferrous ion situated within a tridentate ligand arrangement, termed the triad of His-Asp/Glu-His. In petunia's ACCO, residues His177, Asp179, and His234 constitute this triad. Our sequence alignment suggests that the putative ferrous ion binding site of Kudzu EFE may comprise three conserved amino acids out of the seventeen identified: His189, Asp191, and His268. These amino acids are closely positioned within our conjectured 3D model of EFE (Wu Xu, unpublished data). The Kudzu EFE comprises 10 His residues. Site-directed mutagenesis altering His to Gln yielded activity levels ranging from 0% to 60% compared to wild type. Notably, mutations at His268 and His189—the presumed triad residues—indicated enzyme activities of 0% and 1.8%, respectively. It is likely that H189, D191, and H268 are integral residues forming the triad structure of EFE from the Kudzu strain, with further studies required to test this conjecture.

Reaction Mechanism and Stoichiometry of EFE

Investigations of the EFE reaction utilizing cell-free extracts from P. digitatum and the Kudzu strain have led to the formulation of the following equation for EFE-dependent ethylene production:

AKG + 3 O2 + L-arginine &#;  2 ethylene + succinate + 7 CO2 + guanidine + P5C

With the substrate ratio of 3:1 for AKG and arginine and a product ratio of 2:1:1 for ethylene, succinate, and L-delta 1-pyrroline-5-carboxylate (P5C), Fukuda et al. proposed a unique dual-circuit mechanism wherein EFE catalyzes two distinct reactions in a 2:1 ratio. In the main reaction (two cycles), arginine remains associated as a cofactor as two AKG molecules are transformed into six CO2 and two ethylene. Conversely, in the secondary reaction (one cycle), both AKG and arginine are consumed to produce P5C, guanidine, succinate, and CO2. Although this proposed reaction framework comprises all monitored components identified in these in vitro studies, it only partially aligns with mechanisms established for other enzymes in the superfamily of 2OG/Fe(II)-dependent hydroxylases. The latter reactions involve oxidation of AKG to CO2 and succinate, concurrently coupling with the hydroxylation of a co-substrate such as arginine into hydroxyarginine. To resolve inconsistencies in the EFE reaction mechanism, modern analytical methodologies must be employed. Should the dual-circuit mechanism hold true, does the ethylene-producing catalytic cycle directly link to the succinate-generating cycle, or can EFE act as a promiscuous enzyme facilitating two independent reactions, with the second possibly hydroxylating arginine while concurrently degrading AKG into CO2 and ethylene? If these reactions are indeed separable, opportunities may exist to engineer EFE for ethylene production without the extraneous creation of by-products.

EFE Reaction and Carbon Flow in Cells

While the stoichiometry of the EFE reaction and the proposed dual-circuit mechanism necessitate further validation, they establish a foundational understanding to assess the EFE reaction within a broader metabolic context. To achieve efficient ethylene biosynthesis, metabolic pathways should maximize the molar yield (product:substrate molar ratio) for ethylene while minimizing byproduct production and energy/cofactor consumption. A preliminary elemental node analysis was applied in this context, detailing theoretical yield/energy cost models for ethylene production via common substrates such as CO2 (derived from photosynthesis), glucose, and xylose. The diagrams illustrate the conventional photoautotrophic path for CO2 conversion into ethylene through the Calvin Benson Bassham cycle while examining net carbon uptake alongside ATP and reductant consumption from photosynthetic pathways. It has been established that utilizing xylose or mixtures of sugars enables cogeneration to sustain similar rates of reductant production alongside carbon yields equivalent to glucose (heterotrophic) systems. The methods presented also highlight how routes that employ the CO2-fixing enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) as a non-oxidative pathway can significantly reduce total carbon release in xylose- or glucose-fed systems. Although one-pass flux through Rubisco enhances carbon yields, it incurs the trade-off of increased cofactor needs. Upon reviewing these metabolic processes, it appears that photobiological conversion of CO2 into ethylene is a carbon-negative operation. In contrast, heterotrophic ethylene production from hexosen and pentose sources does not require net cofactor input yet still results in carbon losses, drawing attention toward strategies to recover or minimize lost carbon. An integrated approach utilizing both photosynthetic (potentially multiple passes) and sugar-dependent pathways should lead to enhanced theoretical yields and reduced cofactor requirements. Moreover, conditions that bypass pyruvate dehydrogenase or enhance EFE efficiency could improve carbon yield.

Heterologous EFE Expression and Ethylene Generation

Advancements in metabolic engineering to optimize ethylene production while elucidating metabolic flux pathways toward ethylene are paramount for industrial-scale bioethylene production. Since ethylene production from prevalent metabolites hinges on just one gene (efe), investigating heterologous EFE expression in organisms skilled in utilizing varied feedstocks becomes increasingly relevant. Collectively, ethylene production has been effectively achieved in engineered microorganisms employing sustainable resources like sunlight, cellulose, or glucose derived from biomass.

E. coli and S. cerevisiae

Initial attempts to express heterologous EFE focused on cloning the Kudzu efe gene (featuring its native promoter) into a high-copy pUC19 vector, yielding measurable ethylene production within E. coli. A follow-up effort enhanced EFE yield by employing a lac promoter on a high-copy pUC18 vector or a tac promoter on a medium-copy pBR322 vector, achieving significant results when cultured at a reduced temperature of 25°C. In contrast, raising the culture temperature to 37°C yielded minimal activities, aligning with the decreased half-life (3.3 minutes) of EFE at this temperature; notable changes were also observed in protein localization, resulting in a higher concentration in inclusion bodies. Interestingly, the incorporation of a 15-amino acid peptide from LacZ at the N-terminus in the strain overexpressing EFE helped mitigate inclusion body localization; however, activity levels at 37°C remained low, indicating other factors might also impact EFE activity and stability. Comparative results from various microbial systems indicate the potency of substrate availability as a limiting factor in ethylene production in heterologous expression contexts. When expressed under the constitutive npt promoter on a low-copy plasmid (RS) in E. coli, P. putida, and P. syringae, the P. putida strain yielded the most robust maximum ethylene production. Initial activity was observed early in growth stages, exhibiting a rapid decline, consistent with findings in E. coli strains. Additionally, in vivo EFE activity (with only intracellular substrates) gathered at time points displaying peak production rates were compared against in vitro activities (with exogenously added substrates at saturation), revealing that while wild-type P. syringae demonstrated equivalent in vivo and in vitro activities, the in vitro activities from E. coli; P. putida; and P. syringae overexpressing strains were found to be, respectively, 5-fold, 20-fold, and 40-fold higher than their in vivo counterparts. This highlighted substrate accessibility as a primary limiting factor for in vivo EFE activity. Zhang et al. reported that intracellular AKG levels were maximized during early growth stages, correlating with the observation that AKG can influence levels constraining ethylene synthesis.

Further investigations revealed a tripling of ethylene production in S. cerevisiae when batch cultures undergoing Kudzu efe gene expression switched nitrogen sources from ammonium to glutamate in minimal media (1.0% glucose). This aligns with findings from an in silico modeling study suggesting O2 availability as crucial for optimal ethylene production. More recent analyses in chemostat systems with elevated O2 levels resulted in over 53-fold increases in ethylene production compared to static batch cultures. Although substituting the nitrogen source from ammonium to glutamate improved growth, it yielded no noticeable enhancement in specific ethylene productivity, implying that the growth boost attributed to glutamate addition in batch cultures stemmed from overall cell proliferation instead of heightened EFE efficiency. Surprisingly, adding arginine, the substrate for EFE, led to a more than 50% decline in ethylene productivity, likely redirecting metabolic pathways towards the succinate-forming reaction as postulated by the dual-circuit mechanism.

Cyanobacteria

To connect ethylene generation with the photosynthetic fixation of CO2, the Kudzu efe gene was heterologously expressed in cyanobacteria across various studies. Initial evaluations in Synechococcus elongatus sp. PCC determined that EFE was released using a low-copy pUC303 vector. Notably, unlike E. coli results, the in vivo and in vitro activities showed congruence, suggesting that substrate limitations are not factors in Synechococcus. Various promoters were analyzed for plasmid-based EFE expression, revealing that the native psbA1 promoter showcased the highest activity level, but vectors exhibiting over 100-bp homology to that native region displayed instability—correlated with slow growth rates and lower carbon to ethylene conversion ratios. To address this stability, an integrated approach placing the efe gene at the psbA1 locus in S. elongatus sp. PCC showed promise, resulting in stable ethylene production over 30 generations when associated with a kanamycin resistance gene contextualized behind efe. In cases without kanamycin integration, ethylene rates were reported at four times the level of those exhibiting the integrated resistance gene, achieving comparable rates to some of the highest plasmid-based expression strains. However, these strains still suffered from stress and growth deficits when EFE activity surged, indicating limiting AKG levels might redirect carbon from bilin synthesis thereby leading to noted cellular growth deficiencies.

The Kudzu efe gene was codon-optimized and commercially integrated into the genome of Synechocystis sp. PCC. Stable expression levels were attained leveraging a high-level constitutive pea plant chloroplast psbA promoter when integrating at the slr neutral-site locus. Ongoing research has focused on optimizing the expression of EFE to attain higher ethylene production rates.

Cellulolytic Fungi and Diverse Feedstocks

The pronounced cellulolytic capacities exhibited by many fungi present a promising avenue for producing ethylene from renewable sources. Reports indicate successful integration of the P. syringae pv. glycinea efe gene under a robust cbhI promoter in T. viride, achieving maximal production when utilizing 2.0% cellulose and 0.2% peptone as carbon substrates, with peptone significantly enhancing output. Similarly, another cellulolytic fungus, T. reesei, undertook analyses of heterologous expression of efe from P. syringae pv. glycinea, evaluating numerous promoters to deduce which would yield the highest activity.

P. putida, a gram-negative soil bacterium with a biologically diverse metabolic profile, also offers great potential for synthesizing various compounds through multiple waste feedstocks. Wang et al. designed a vector to integrate multiple copies of efe from P. syringae pv. glycinea into sites within the 16S rDNA of P. putida. Utilizing this engineered construct effectively increased ethylene production rates and glucose-to-ethylene conversions, achieving the most competitive reported production rates for both native and recombinant organisms.

Harvesting Biologically Produced Ethylene

Critical considerations concerning the biological production of ethylene include effective heat and gas harvesting methods. In typical petrochemical contexts, cryogenic distillation is employed to isolate ethylene from gaseous mixtures—an energy-consuming endeavor but capable of retrieving numerous gaseous outputs. Alternative methodologies encompass solvent extraction, pressure swing adsorption through zeolites, and membrane separations. Special protocols need to be formulated for capturing biologically derived ethylene based on gas mixture compositions, which may inherently include components such as CO2, moisture, nitrogen, and oxygen. In photosynthetic systems where O2 is co-generated with ethylene, safety concerns regarding ethylene's combustibility arise (notably in concentrations from 2.7% to 36% v/v). Targeted engineering designs aim to mitigate these risks, while biologically produced ethylene is expected to lack the metals and additional contaminants often prevalent in fossil fuel-sourced ethylene supplies, potentially establishing it as a preferred alternative for high-purity chemical productions and clean fuels.

Prospective Research Directions

The realm of bioethylene technology remains in preliminary stages, though significant advancements across numerous research domains are required to facilitate a shift from fossil-derived feedstocks. Current efforts include technoeconomic assessments of bioethylene production within cyanobacterial frameworks, aimed at dictating future research trajectories. As noted, foundational comprehension surrounding EFE’s structure, functionality, and reaction dynamics is insufficient. A systematic examination of EFE and its related sequences and structures to delineate conserved elements alongside a conjectured catalytic active site will enhance understanding toward the dual-circuit catalytic mechanism proposed. Structural elucidation of EFE should further provide insight into optimizing protein engineering for better carbon yield and thermal stability. Additionally, precise reaction stoichiometry aligned with carbon flux evaluations will support metabolic pathway engineering toward formulating efficient production routes.

The integration of synthetic biology could fast-track strain development by meticulously designing pathways aimed at enhancing ethylene production yield. To fully harness synthetic biology's potential, tools for high-throughput screening/selection are essential for monitoring ethylene levels and its precursors. Genetically encoded sensing systems for substrates like AKG and arginine have already been developed, paving the way for potential ethylene sensor designs leveraging ethylene receptors identified in both plants and cyanobacteria.

Ultimately, the scaling up of production will require the establishment of cost-effective bioreactors equipped with improved oxygen transfer capabilities for non-photosynthetic systems and efficient light delivery for photosynthetic establishments, alongside creating customized harvesting methodologies designed for the particularities of biologically generated gas streams.

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