Acetone-butanol-ethanol Research Articles

Techno-Economic Analysis of Upgrading Corn Stover-Based Acetone, n-Butanol, and Ethanol to Higher Ketones and Alcohols: Fuels or Fine Chemicals?

Acetone–butanol–ethanol (ABE) fermentation is a traditional industrial exploitation with a history of 100 years and the second largest fermentation industry in the world, second only to bioethanol fermentation. However, low ABE concentration in the fermentation broth, a complex separation process, and high separation cost are the bottlenecks of the biobutanol industry. Therefore, developing an efficient and energy-saving ABE upgrading process gives another approach to the reindustrialization of ABE fermentation, even with the completeness of fossil resource-based products. Here, we report on a coupling salting-out catalytic process intensification technique in which acetone was alkylated with ethanol and 1-butanol by Pd/C + K3PO4, and the higher ketone and alcohol products were separated by the salting-out effect of K3PO4. The higher ketones and alcohols were experimentally obtained with >70% mass fractions at 200–250 °C under the fermentation-derived H2 atmosphere and can be used as fuel precursors or fine chemicals. Aspen Plus was employed to simulate corn stover-based biobutanol production and the upgrading process through a series of analyses on raw materials cost, capital cost, operating cost, product selling prices, net present values, and sensitivities. A comparison between fuel precursors and fine chemicals was carried out, and fine chemicals turned out to be the preferred choice if the selectivity of the products and further purification process could be optimized. The results of a sensitivity analysis show that the ABE concentration process is one of the most energy-intensive processes. Reducing the costs of raw materials is one strategy to improve the completeness of ABE fermentation.

Production of butyl butyrate from lignocellulosic biomass through Escherichia coli-Clostridium beijerinckii G117 co-culture

Butanol toxicity, oxygen sensitivity, and high substrate cost limit wider applications of the traditional Clostridium acetone-butanol-ethanol (ABE) fermentation. In this study, a wild-type Clostridium beijerinckii strain was partnered with an engineered Escherichia coli strain to improve the ABE fermentation through a co-culture. Butanol and butyrate produced by C. beijerinckii were converted into butyl butyrate – a product that can be in situ removed to reduce product toxicity – by expressing a CoA transferase and an alcohol acyltransferase in E. coli. When integrated with a pretreatment technology and using commercial cellulase, the co-culture produced 1280 mg/L butyl butyrate from rice straw in bioreactors, without the need of maintaining a strict anaerobic condition. As a process harnessing both the genetic tractability of E. coli and the superior acid- and alcohol-producing ability of Clostridium, this co-culture fermentation will be useful in producing valuable esters from low-cost waste streams.

Formate as a supplementary substrate facilitates sugar metabolism and solvent production by Clostridium beijerinckii NCIMB 8052

Microbial utilization and conversion of organic one-carbon compounds, such as formate and methanol that can be easily produced from CO2, has emerged as an attractive approach for biorefinery. In this study, we discovered Clostridium beijerinckii NCIMB 8052, a typical solventogenic Clostridium strain, to be a native formate-utilizing bacterium. 13C isotope analysis showed that formate could be metabolized via both assimilation and dissimilation pathways in C. beijerinckii NCIMB 8052. Notably, the use of formate as the supplementary substrate by this strain could significantly enhance its glucose consumption and ABE (acetone-butanol-ethanol) production, largely due to the up-regulation of genes responsible for glycolysis and glucose transport under formate stress. Based on these findings, we further improved formate tolerance of C. beijerinckii NCIMB 8052 by adaptive laboratory evolution, generating an evolved strain Cbei-FA01. The Cbei-FA01 strain could produce 23.0 g/L of ABE solvents using glucose and formate as dual substrates, ∼50% higher than that of the wild-type strain under the same condition. Moreover, such a promotion effect of formate on ABE production by Cbei-FA01 was also observed in fermenting a glucose-xylose mixture. This work reveals a previously unreported role of formate in biological ABE production, providing a new approach to utilize this one-carbon source.

Moving towards the Application of Biocatalysis in Food Waste Biorefinery

Waste valorization is an important strategy to reduce environmental pollution and dependency on petroleum-based fuels. In this regard, utilization of food waste as a versatile and low-cost resource is important. Several advanced catalytic methods for the valorization of food waste have been widely investigated for the production of liquid biofuels. Along this line, chemical catalysts have been explored for the synthesis of liquid biofuels. Chemo-catalysis is mainly metal based, which requires harsh process conditions. Alternatively, biocatalysts are currently being investigated as a result of several advantages such as mild reaction conditions, recyclability, selectivity and biodegradability. In this work, recent biocatalytic technologies for the preparation of liquid biofuels through food waste valorization are discussed thoroughly. Lipases are employed for the synthesis of biodiesel and the upgradation of bio-oil, whereas methane mono-oxygenases could be explored for the production of methanol via the oxidation of methane generated from food wastes. Industrial production of ethanol from food waste using bioconversion technologies is a success story. To date, there has been no specific report on the use of food waste for propanol preparation using enzymes. The ABE process (Acetone–Butanol–Ethanol) (using suitable microorganisms) is used for butanol preparation, where the vacuum stripping system is integrated to remove butanol from the broth and circumvent inhibition. The synthesis of hydrocarbon fuels from fatty acids and triglycerides can be carried out using enzymes, such as carboxylic acid reductase and fatty acid photodecarboxylase (an algal photoenzyme). Both carboxylic acid reductase and fatty acid photodecarboxylase have not yet been applied in the direct valorization of food wastes. Furthermore, limitations of the reported methods, societal and economic aspects and a fresh perspective on the subject, along with important examples, are described.

Efficient co-production of fermentable sugars and biobutanol from corn stover based on a novel butyric acid pretreatment strategy

Efficient deconstruction of lignocellulosic biomass by pretreatment is important for the production of fermentable sugars and biofuels. This study used butyric acid as a novel catalyst for corn stover pretreatment. The effects of butyric acid pretreatments on chemical composition and enzymatic digestibility were systematically investigated. The results indicate that a high linear correlation between xylan removal and fermentable sugars yield (r2 = 0.91) was found. Pretreatment of corn stover with 50 g/L butyric acid at 180 °C for 0.5 h removed 83.17% of xylan and recovered 86.99% of glucan, and fermentable sugars yield of pretreated corn stover reached 97.16%. The physiochemical characterization results show that crystalline index (CrI) of corn stover was increased from 30.13% to 45.04%−48.38% after pretreatments, and the chemical structure and morphological features also exhibited positive changes for enhancing enzymatic hydrolysis. Meanwhile, the liquid fraction of pretreated slurry can be used for recycled pretreatment with 73.81% of fermentable sugars yield after four cycles. Additionally, the residual butyric acid was re-utilized as the co-substrate for Clostridium acetobutylicum ATCC 824 to produce biobutanol via acetone-butanol-ethanol (ABE) fermentation. The final production of butanol and solvents reached 8.38 g/L and 12.63 g/L under an optimized intermittent feeding strategy with a higher butanol/acetone ratio of 2.40 g/g. Overall, this work presents a novel pretreatment strategy to co-produce fermentable sugars and advanced biofuel from abundant lignocellulosic biomass.

Co-fermentation of lactic acid and acetone-butanol-ethanol (ABE) from the deep eutectic solvent–pretreated Aloe vera leaf rind through sequential valorization of holocellulose

Co-fermentation of lactic acid and acetone-butanol-ethanol (ABE) from the deep eutectic solvent–pretreated Aloe vera leaf rind through sequential valorization of holocellulose

Model-driven approach for the production of butyrate from CO2/H2 by a novel co-culture of C. autoethanogenum and C. beijerinckii.

One-carbon (C1) compounds are promising feedstocks for the sustainable production of commodity chemicals. CO2 is a particularly advantageous C1-feedstock since it is an unwanted industrial off-gas that can be converted into valuable products while reducing its atmospheric levels. Acetogens are microorganisms that can grow on CO2/H2 gas mixtures and syngas converting these substrates into ethanol and acetate. Co-cultivation of acetogens with other microbial species that can further process such products, can expand the variety of products to, for example, medium chain fatty acids (MCFA) and longer chain alcohols. Solventogens are microorganisms known to produce MCFA and alcohols via the acetone-butanol-ethanol (ABE) fermentation in which acetate is a key metabolite. Thus, co-cultivation of an acetogen and a solventogen in a consortium provides a potential platform to produce valuable chemicals from CO2. In this study, metabolic modeling was implemented to design a new co-culture of an acetogen and a solventogen to produce butyrate from CO2/H2 mixtures. The model-driven approach suggested the ability of the studied solventogenic species to grow on lactate/glycerol with acetate as co-substrate. This ability was confirmed experimentally by cultivation of Clostridium beijerinckii on these substrates in batch serum bottles and subsequently in pH-controlled bioreactors. Community modeling also suggested that a novel microbial consortium consisting of the acetogen Clostridium autoethanogenum, and the solventogen C. beijerinckii would be feasible and stable. On the basis of this prediction, a co-culture was experimentally established. C. autoethanogenum grew on CO2/H2 producing acetate and traces of ethanol. Acetate was in turn, consumed by C. beijerinckii together with lactate, producing butyrate. These results show that community modeling of metabolism is a valuable tool to guide the design of microbial consortia for the tailored production of chemicals from renewable resources.

Pretreatment with a combination of steam explosion and NaOH increases butanol production of enzymatically hydrolyzed corn stover

As an essential lignocellulosic source, corn stover (CS) arouses an extensive interest in transforming into bio-butanol and high-value-added products. Optimization experiments of medium and kinetic fermentation experiments of Clostridium acetobutylicum CICC 8008 were conducted. The CS was pretreated by a combination of steam explosion and NaOH, followed by saccharification by cellulase. The saccharification liquor contained 65.91 g/L of total reducing sugar. The optimum fermentation medium was 1,000 mL saccharification liquor supplemented with 4.89 g/L yeast extract, 6.9 g/L CaCO3, and 10.56 mg/L biotin. When the optimum medium was used in the butanol fermentation process, the maximum acetone-butanol-ethanol (ABE) content was 41.78 g/L, and the contents of acetone, butanol, and ethanol were 9.73, 23.82, and 8.23 g/L, respectively. The maximum specific growth rate was 0.066 h−1, the coefficient of growth was 2.29 g/(gL), and no substrate and production inhibition were found. ABE was the growth-associated metabolite. Collectively, our current findings provided valuable insights into the efficient application of lignocellulose biomass into the biorefinery of butanol.

A novel integrated process for selective preparation of jet-range fuels from low carbon alcohols and ABE

The development of effective route for the production of jet fuels from low carbon alcohols or ABE (acetone/butanol/ethanol) is of great significance for reducing carbon emission. The purpose of this work is to prove that jet fuels can be selectively produced from ABE and lower alcohols. This novel synthetic strategy was based on an integrated process: alcohols dehydration to light olefins over the SAPO-34 catalyst and olefin polymerization to jet fuels over the 2%Ni@5%Fe@Hβ catalyst. The comparison between the single bed reaction mode and double bed reaction mode, the active sites of the catalysts, and the reusability of the catalysts were investigated. The double bed reaction mode significantly improved the selectivity of jet fuels in the catalytic conversion of ABE and low carbon alcohols. High selectivity of jet-range fuels (80.1%) and high conversion of ABE (94.8%) were achieved by the integrated process.

Economic analysis of biobutanol recovery from the acetone-butanol-ethanol fermentation using molasses

Commercial production of biobutanol via acetone-butanol-ethanol (ABE) fermentation is challenged by the energy-intensive recovery of biobutanol from inherently diluted fermentation broth. Towards the solution of this problem, four combinations (scenarios) of separation techniques such as distillation, gas stripping (GS), liquid-liquid extraction (LLE), and pervaporation (PV) are investigated for the minimum unitary production cost of biobutanol. Cost components for the installation and operation of the plant are estimated using Matlab software and method given by Peters and Timmerhaus. Production costs with significant variables viz. biobutanol yield, price of molasses, and fermenter volume are individually estimated. Gas stripping followed by distillation is found as the most economical scenario with a unitary production cost of INR 74/kg. The optimum values of yield and fermenter volume are found as 0.4 kg ABE/kg sugars and 100 m3, respectively. The optimization of three major variables around the baseline values provides nearly 10% reductions in the cost with present fermentation technology. This economic analysis of biobutanol recovery certainly contributes to the commercial production of biobutanol from renewable raw materials. The economic feasibility of biodiesel production may offer better environment to the mankind.

Biobutanol production from cocoa pod husk through a sequential green method: Depectination, delignification, enzymatic hydrolysis, and extractive fermentation

The effect of various treatments on the production of reducing sugar and biobutanol from cocoa pod husk (CPH) through depectination, delignification, enzymatic hydrolysis, and fermentation was studied to obtain the best strategy for sequential processes to valorize CPH to biobutanol. Surfactants of Tween 80, Polyethylene Glycol (PEG) 6000, and Sodium Dodecyl Sulfate (SDS) was added to enhance the performance of enzymatic hydrolysis, and L-cysteine addition to support fermentation by Clostridium saccharoperbutylacetonicum N1–4. The optimum activity of the cellulase was at 0.25 FPU/g. Depectination, microwave-assisted delignification, and 2.5 g/L PEG 6000 addition sequence produced the highest sugar concentration of 9.24 g/L. The highest butanol and acetone-butanol-ethanol (ABE) concentrations of 20.4 g/L and 54.4 g/L, respectively, were obtained using immobilized cells and L-cysteine supplementation. ABE production by extractive fermentation was enhanced using immobilized cells and L-cysteine supplementation.

Waste-to-fuel technologies for the bioconversion of carrot discards into biobutanol

Carrot discard was evaluated as a raw material for acetone-butanol-ethanol (ABE) fermentation. Different strategies based on hydrothermal pretreatment and/or enzymatic hydrolysis were compared for biobutanol production from carrot discard pulp. In addition, the use of different types of enzymes and diverse enzyme mixtures were evaluated. In this way, total sugar recoveries of up to 76%, and butanol and ABE concentrations of 7.4 and 11.5 g/L, respectively (74 g butanol and 115 g ABE/kg carrot pulp), were achieved when the carrot discard pulp was enzymatically hydrolyzed, without pretreatment, using a mixture of enzymes of Cellic CTec2 and Viscozyme L at a dosage of 0.1 and 0.2 g/g, respectively. When a hydrothermal pretreatment was applied, a total sugar recovery of 88%, 6.9 g/L butanol and 10.1 g/L ABE (69 g butanol and 101 g ABE/kg carrot pulp) were attained using the same mixture of enzymes. In this way, no hydrothermal pretreatment would be necessary to produce ABE from carrot discard, which is very interesting for the profitability of the process. Furthermore, the carrot discard juice yielded 6.4 and 9 g/L butanol and ABE, respectively, showing that all the carrot discards could be used for ABE production.

Mechanistic modeling of redox balance effects on the fermentation of eucalyptus wood-derived xylose to acetone-butanol-ethanol

A Michaelis-Menten-like kinetic model is proposed to simulate the effects of redox balance on Clostridium beijerinckii NCIMB 8052 metabolism during fermentation of biomass-derived xylose to acetone-butanol-ethanol (ABE). The model builds on previous metabolic kinetic models by introducing six mass balance differential equations for the redox pairs NADH/NAD+, NADPH/NADP+, and oxidized/reduced ferredoxin. Batch cultures were conducted using laboratory-grade or eucalyptus-derived xylose, and kinetic parameters were estimated by minimizing the difference between experimental (substrate, metabolites, and cell) and model results using a genetic optimization algorithm. The simulations agreed well with experimental data, and a sensitivity analysis showed that the rate of reoxidation of reduced ferredoxin used for NADPH regeneration is the most relevant reaction for the synthesis of acids and ABE. Furthermore, an increase in NADPH consumption due to the reduction of eucalyptus-derived furfural to furfuryl alcohol was only significant for the production of butanol. Differences in the values of the Michaelis-Menten constants, Km’s, depending on the xylose source, suggest that a decrease in the conversion rates of lactate to pyruvate and acetate to acetyl-CoA considerably affected the biosynthesis of butanol from eucalyptus-derived xylose. The proposed model was effective in assessing the effects of redox balance on ABE fermentation, suggesting its use for the design of metabolic engineering strategies to improve butanol synthesis from furfural-containing biomass hydrolysates.

Carbonized biomass as an immobilization carrier in acetone-butanol-ethanol (ABE) fermentation by Clostridium beijerinckii JCM 8026

Carbonized biomass as an immobilization carrier in acetone-butanol-ethanol (ABE) fermentation by Clostridium beijerinckii JCM 8026

Construction and metabolic analysis of acetone-butanol-ethanol fermentation using mixed acetic acid and lactic acid in wastewater

To reuse organic acids in wastewater, efficient acetone-butanol-ethanol (ABE) fermentation was constructed using mixed acetic acid and lactic acid. The mixed acids were co-consumed with glucose, producing higher butanol concentration (6.96 g/L) and butanol yield (0.494 C-mol/C-mol) than using each single acid (6.22 g/L and 0.432 C-mol/C-mol). This result was verified by 13 C tracer experiment that addition of lactic acid improved carbon distribution to butanol instead of acetone , while acetic acid was readily consumed than lactic acid at initial fermentation stage. Subsequently, pH-stat fed batch fermentation at low pH of 5.5 further improved acid consumptions (6.34 g/L acetic acid and 9.06 g/L lactic acid) and solvent productions (12.4 g/L butanol and 6.50 g/L acetone). Finally, ABE fermentation using wastewater of lactic acid fermentation was performed without inhibition. Thus, efficient ABE fermentation can be implemented using mixed acetic acid and lactic acid, which is superior to using single acid. • This is the first study using mixed acetate and lactate for ABE fermentation. • 13 C experiment verified the metabolic flux to butanol was improved by the acids. • pH and glucose played an important role in acid consumption and solvent production. • Fermentation from lactic acid fermentation broth was performed without inhibition.

Identification of serine/threonine kinases that regulate metabolism and sporulation in Clostridium beijerinckii.

Serine/threonine protein kinases (STKs) are important for signal transduction and involved in multiple physiological processes, including cell growth, central metabolism, and sporulation in bacteria. However, the role of STKs in solventogenic clostridia remains unclear. Here, we identified and comprehensively investigated six STK candidates in Clostridium beijerinckii. These STKs were classified into four groups with distinct characteristics via analysis of genetic organizations, prediction of protein domains, and multiple sequence alignment. Cbei0566 is a member of the PrkA family with 41% identity to PrkA from Bacillus subtilis, and both Cbei0666 and Cbei0813 are two-component-like STKs. Cbei1151 and Cbei1929 belong to the Hanks family STKs and consist of a cytoplasmic catalytic domain, a transmembrane region, and extracellular sensor domains. In-frame deletion mutants of cbei0566, cbei0666, cbei1929, and cbei2661 displayed similar cell growth with wild type. Both Δcbei0666 and Δcbei2661 improved acetone-butanol-ethanol (ABE) production by 14.3% (19.2g/L vs. 16.8g/L), and the sporulation frequencies of Δcbei0566, Δcbei1929, and Δcbei2661 significantly decreased to 35.5%, 55.1% and 44.8%, respectively. The restored phenotypes after genetic complementation demonstrated their direct link to STKs deletion. Remarkably, overexpressing cbei0566 contributed to 41.5% more spore formation and cbei1929 overexpression enhanced ABE production from 19.3 to 24.2g/L, along with 25% less acids. These results revealed that Cbei0566 and Cbei1929 had prominent regulatory functions. This study expands the current knowledge of the existence and functions of STKs in prokaryotes and highlights the importance of STK-mediated signaling networks in developing superior strains. KEY POINTS: • First reported serine/threonine protein kinases in solventogenic clostridia • Six STKs with distinct properties possessed diverse functions in C. beijerinckii • Cbei1929 and Cbei0566 remarkably regulated solventogenesis and sporulation.

Effects of Acetone-Butanol-Ethanol (ABE) addition on HCCI-DI engine performance, combustion and emission

Recent research has shown the potential of long-chain alcohols such as butanol for biofuel. However, the production quantity of butanol is very low and consumes high energy in its purification process. To solve such a problem, one of the effective approaches is to use acetone-butanol-ethanol (ABE) directly from the fermentation process. This study aims to investigate the effects of ABE-diesel blends in an HCCI-DI engine. It was found that although the presence of oxygen in ABE could theoretically promote complete combustion and eventually decrease HC and CO emissions, other significant factors may have played more dominant roles in affecting the HC and CO emissions. The experimental investigation on an HCCI-DI engine fuelled with ABE also did not reduce PM and Soot emissions. Furthermore, the peak in-cylinder combustion pressure decreased with fluctuating heat release rate. However, their p-V areas were larger than pure diesel fuel, which implied a higher indicated work produced per cycle. The addition of ABE also successfully decreased CO2 and NOx emissions. Moreover, improved engine performance in terms of higher BTE, lower BSFC and EGT were observed. All in all, considering its satisfying improved engine performance; ABE has the potential to become a promising alternative biofuel.

Selection of protic ionic liquids for the improved production of butanol from rice straw

Biomass pretreatment with protic ionic liquids (PILs) has been proved as an economically attractive method to enhance saccharification yield. This work evaluated the use of six PILs -based on acetate and hydrogen sulfate anions - using water as cosolvent for processing a high solid loading of rice straw (RS). Among the investigated PILs, the most promising results were obtained by 2-hydroxyethylammonium acetate [MEOA][OAc] containing 20 %w/w water, achieving 87.3 % of glucose and 68.6 % of xylose yield, respectively, and 98.3 % of delignification at 150 °C for 2 h with 15 %w/w solid loading. Five recovery and reuse cycles were successfully performed with [MEOA][OAc], obtaining a delignification degree of 69.5 % after the fifth reuse. Process viability was evaluated by enzymatic hydrolysis followed by acetone-butanol-ethanol (ABE) fermentation using Clostridium beijerinckii. Similar saccharification yields (89.9 ± 6.8 % glucose and 71.5 ± 4.7 % xylose) and butanol production (7.3 ± 0.1 g/L, 81.7 ± 1.7 g butanol per kg of RS) were achieved for fresh and recycled [MEOA][OAc], demonstrating the feasibility of PIL reuse. This study showed the potential of [MEOA][OAc] as a cost-effective pretreatment agent for rice straw valorization into an advanced biofuel such as butanol.

Efficient Cross-Coupling of Acetone with Linear Aliphatic Alcohols over Supported Copper on a Fluorite-Type Pr2Zr2O7

In cross-coupling of biomass-derived acetone and alcohols contributing to the production of carbon-elongated chemicals and fuels, the essential catalyst components are metal dispersion for alcohol dehydrogenation and, more importantly, basicity for carbon–carbon coupling. Herein, we report the potential of co-precipitated praseodymia–zirconia solid solution (Pr2Zr2O7) as a support of Cu catalyst for the conversion of acetone and butanol into C7 and C11 products. Cu/Pr2Zr2O7 exhibits a high yield of C7 and C11 (ca. 84%) compared to Cu/ZrO2 and Cu/PrO1.83. Moreover, it is robust under the employed solvent-free conditions owing to a solid solution of Pr2Zr2O7 compared to PrO1.83 showing phase transition to PrOHCO3. It is also tolerant to up to 5 wt % water of the reactant mixture, recyclable once adequate post-treatment is employed after the reaction, and can convert the acetone–butanol–ethanol mixture into C5–C11 products at the nearly equivalent yield (82%) to the acetone–butanol mixture. Therefore, the Cu/Pr2Zr2O7 reported herein is an efficient catalyst for the coupling of acetone with linear aliphatic alcohols into biofuel precursors.

Combination of pH adjusting and intermittent feeding can improve fermentative acetone-butanol-ethanol (ABE) production from steam exploded corn stover

Steam explosion (SE) is an effective and industrially scalable lignocellulose pretreatment method that can be used for second-generation acetone-butanol-ethanol (ABE) fermentation. Nevertheless, a series technical barrier including the high inhibitors content in hydrolysate, and low concentration and low yield of ABE production always hindered the availability of the SE technique. In this study, aiming to solve the above obstacles and boosting cellulosic ABE production, the ABE fermentation process was intensified by a novel fed-batch strategy that combined the pH adjusting with intermittent feeding. Results revealed that maintaining the pH at 6.5 for initial 12 h of cultivation ensures the smoothly shifting of the acidogenesis phase to solventogenesis phase of the C. acetobutylicum cells, so that avoided the acid crash. While the lag period of fermentation was obvious shorted by intermittent feeding the concentrated SE hydrolysate into the dilute broth. Consequentially, 17.75 g/L of ABE (containing 11.75 g/L of butanol) can be obtained after 48 h of inoculation, with the yield and productivity of ABE were 0.36 g/g (of sugars) and 0.37 g/L‧h, respectively. Correspondingly, 194.9 g of ABE (including 129.0 g of butanol) can be produced from 1 kg of dried corn stover. Therefore, the novel process intensifying method could obviously improve the biobutanol production, which showed high potential and applicability in large-scale ABE productions.