- Open Access
Synthesis and techno-economic assessment of microbial-based processes for terpenes production
© The Author(s) 2018
- Received: 1 May 2018
- Accepted: 9 October 2018
- Published: 27 October 2018
Recent advances in metabolic engineering enable the production of chemicals from sugars through microbial bio-conversion. Terpenes have attracted substantial attention due to their relatively high prices and wide applications in different industries. To this end, we synthesize and assess processes for microbial production of terpenes.
To explain a counterintuitive experimental phenomenon where terpenes such as limonene (normal boiling point 176 °C) are often found to be 100% present in the vapor phase after bio-conversion (operating at only ~ 30 °C), we first analyze the vapor–liquid equilibrium for systems containing terpenes. Then, we propose alternative production configurations, which are further studied, using limonene as an example, in several case studies. Next, we perform economic assessment of the alternative processes and identify the major cost components. Finally, we extend the assessment to account for different process parameters, terpene products, ways to address terpene toxicity (microbial engineering vs. solvent use), and cellulosic biomass as a feedstock. We identify the key cost drivers to be (1) feed glucose concentration (wt%), (2) product yield (% of maximum theoretical yield) and (3) VVM (Volume of air per Volume of broth liquid per Minute, i.e., aeration rate in min−1). The production of limonene, based on current experimental data, is found to be economically infeasible (production cost ~ 465 $/kg vs. market selling price ~ 7 $/kg), but higher glucose concentration and yield can lower the cost. Among 12 terpenes studied, limonene appears to be the most reasonable short-term target because of its large market size (~ 160 million $/year in the US) and the relatively easier to achieve break-even yield (~ 30%, assuming a 14 wt% feed glucose concentration and 0.1 min−1 VVM).
The methods proposed in this work are applicable to a range of terpenes as well as other extracellular insoluble chemicals with density lower than that of water, such as fatty acids. The results provide guidance for future research in metabolic engineering toward terpenes production in terms of setting targets for key design parameters.
- Process systems engineering
- Process simulation
- Microbial production
- Vapor liquid equilibrium
- Biphasic fermentation
- Fatty acids
Recent advances in metabolic engineering enable the use of microbes such as E. coli and S. cerevisiae for the production of chemicals [1–12]. Compared to traditional fossil fuel-based processes, bio-processes can be advantageous for their mild production conditions and good selectivity toward a specific product . Also, the chemicals can be produced directly using microbes instead of being converted via multiple conversion steps (some of which can have low yield and high cost) from fossil fuel feedstocks.
Terpenes (also known as isoprenoids or terpenoids) are a class of organic compounds biosynthetically derived from isoprene (C5H8: CH2=C(CH3)–CH=CH2) and can be classified into groups according to the number of carbons they contain: hemiterpene (C5, i.e., isoprene), monoterpenes (C10; major interest of many studies), sesquiterpenes (C15), diterpenes (C20), triterpenes (C30), etc. [14–17]. Terpenes have been attracting substantial attention due to their relatively high prices and wide applications in chemical, food, cosmetics, pharmaceutical, fragrance, flavor and biotechnology industries [14, 18–23]. Some terpenes, such as limonene and linalool, are also potential drop-in biofuels and platform chemicals to produce other value-added products [24–26].
The microbial conversion of terpenes from sugar via microbes has been reviewed extensively [14, 27–36]. Terpenes can be produced mainly via the mevalonate pathway, or 1-deoxy-d-xylulose-5-phosphate (DXP) pathway. Most terpenes are produced extracellularly, and they are insoluble and lighter (in terms of density) than water, thus forming a top oil phase in the liquid fermentation broth. Note that we do not consider the special case where terpenes fail to form a separate phase from water due to the presence of surface active impurities in the broth. Recent studies indicate that the fermenter can be tuned to favor globule coalescence and thus the formation of a separate phase [37–39]. Also, microbial production of terpenes has demonstrated high selectivity toward a specific product . Although intracellular components (such as amino acids and nucleoids) will be released after cell death, compounds that are insoluble and lighter than water (as the terpene product is), mainly lipids, are actually bound to the cell membrane debris and settle to the bottom, and are thus naturally separated from the product (on the top). Therefore, downstream separation cost tends to be relatively low. In addition, several terpenes (such as limonene and pinene) were estimated to have the potential to reach a promising profit margin in a recent study that identifies economically promising bio-based chemicals .
US market data and properties of selected terpenes
Market price ($/kg)
Market volume (106 kg/year)
Market size (106 $/year)
Max. yield (g product/g glucose)
Solubility (g/L, 25 °C)
Boiling point (°C, 1 atm)
Microbial production of limonene poses great potential in addressing such issues and thus has been the focus of many studies [16, 48, 52–61]. Several downstream separation methods have also been reported on laboratory scale, including culture extraction, solvent overlay, solid-phase micro-extraction, adsorbent polydimethylsiloxane bar, and continuous headspace removal using a cold trap [43, 60, 62–65]. However, studies on large-scale separation process synthesis and assessment of the entire production process have been limited . In addition, a systematic analysis on a counterintuitive experimental phenomenon is still lacking: terpenes such as limonene are often found to be 100% present in the vapor phase after bio-conversion, despite limonene’s normal boiling point being 176 °C and the reactor operating temperature being only ~ 30 °C. Therefore, in this work, we analyze the vapor–liquid equilibrium for systems containing terpenes, synthesize alternative processes for microbial terpenes production, and perform techno-economic assessment, thereby identifying major cost drivers and key insights for all alternative process configurations.
The outline of this paper is as follows. In the “Methods” section, we discuss the bio-conversion process and analyze the vapor–liquid equilibrium and its implications on downstream separations. In the “Results and discussion” section, we present three process configurations, which are demonstrated using limonene and perform economic assessment. This is followed by an expanded study, where the costs and the corresponding process configurations in the entire feasible space, defined in terms of three key process parameters, are analyzed. Finally, we generalize our discussion to account for other terpenes, microbes, bio-conversion systems, ways to address terpene toxicity on microbes, and cellulosic biomass as a feedstock.
The entire microbial terpene production process consists of upstream bio-conversion and downstream separations. We assume 40 T/h (“T” = metric ton) glucose supplied to the bio-conversion system (which can involve multiple fermenters in parallel), where a terpene product is produced by a microbe such as E. coli or S. cerevisiae. We choose a rate of 40 T/h because this is the amount of sugar produced by hydrolyzing 2000 T/day biomass in the NREL study , which we use as the basis of our bio-refinery capacity. Our goal is to obtain a technical grade terpene product (e.g., ≥ 95 wt% for limonene) after downstream separations.
The supply of nitrogen and phosphorus nutrients (e.g., using diammonium phosphate) for cell growth is neglected here because the cost is negligible (less than 1% of the total operating cost in the NREL study). Also, we assume continuous operation of the fermenter (i.e., chemostat). Experimental data obtained based on batch or fed-batch reactors (e.g., in kg) are converted into the equivalent data for chemostats (e.g., in kg/h) using the methods discussed in “Methods” section of Additional file 1.
The simulation of the entire process, including downstream separation; mass and energy balance calculations; and economic assessment, is all performed in SuperPro Designer  with built-in techno-economic parameters (see the specific values in Additional files 1 and 2). The split fractions calculated based on the discussed VLE calculation methods (see implementation in Additional file 2) are imported as fixed parameters into SuperPro to help specify the component flowrates after fermentation.
Vapor–liquid equilibrium (VLE) analysis
VLE analysis in the fermenter
VLE analysis in the condenser
To maximize product recovery, we operate the condenser such that λ = 100% (all the product is condensed), and the condenser temperature TC can be calculated accordingly (see Sect. 5 of Additional file 1). For limonene, we calculate TC ≅ 0 °C.
Parameters assumed for the three case studies
Glucose concentration (wt%)
Yield (g product/g glucose)
Yield (% of max.)
Residence time (h)
VLE implications on downstream separation
Alternative process configurations
Economic assessment of the case studies
Unlike many other bio-based chemical production processes, where separation accounts for 60–80% of the total cost [13, 79], separation is not the major cost driver here mainly because limonene (and most other terpenes) is extracellular, insoluble, and lighter than water (in terms of density), which requires a simple centrifugal decantation. Note that the separation cost will increase when a product’s oil globule size is smaller and the density is closer to that of water (based on Stokes’s law), which can lead to a smaller globule rising velocity in the decanter and thus larger equipment. For example, if we consider a terpene with globule diameter 1 μm and density 950 g/L, instead of limonene (20 μm and 841 g/L), then the centrifugation cost in Case 3 will increase by 40% (although the total cost will increase by just 0.1% due to high feed glucose cost).
General economic assessment
It is also worth noting that evaporation of limonene actually generates a more concentrated product stream after condensation (e.g., 26 g/L after condensation vs. 0.22 g/L assuming no evaporation in the fermenter in Case 1; see Fig. 7a), which facilitates downstream separation. However, we should not increase VVM to “push” more product to the vapor phase, because a higher VVM for each glucose concentration and yield combination in Fig. 10 leads to a higher cost. In other words, the additional costs of compression and condensation due to increased VVM outweigh the savings from separation (centrifugal decantation).
Addressing toxicity: use of solvent vs. microbial engineering
Specifically, we adopt the parameters from Case 3 (glucose concentration = 24 wt%, yield = 95%, and VVM = 0.01 min−1) and add dodecane as a solvent (miscible with limonene and immiscible with water) into the fermenter. Since dodecane prevents evaporation, only the liquid stream is treated with centrifugal decantation. The limonene–dodecane mixture is then distilled to obtain the final product, while dodecane is recycled. In terms of the amount of dodecane used, values between 5 and 20 vol% are reported in the literature [54, 60, 80, 81]. We assume 10 vol% here.
The method presented in this work can be used to study systems producing different terpenes (by modifying Eq. 2 and product physical properties accordingly), as well as different microbes (by modifying Eq. 1) and bio-conversion systems (by replacing the fermenter with, for example, an open pond, and glucose with CO2 to account for photosynthesis instead of fermentation).
Minimum break-even yields and the corresponding optimal process configurations for 12 terpenes
Minimum break-even yield (% max.)
Minimum break-even yields and the corresponding optimal process configurations for 12 terpenes
Minimum break-even yield (% max.)
Limonene appears to be the most promising short-term target because it has a comparatively high expected profit based on our current assumptions (Table 3), as well as a relatively low break-even yield (Table 4). For the terpenes with high prices yet low market volumes, the break-even yields are even easier to achieve, but the total market size is small. More accurate market volume data are needed to better quantify their economic prospect. Also, low-volume products are often used for niche markets (e.g., cosmetics and pharmaceuticals), which have much stricter final product requirements, and thus their actual costs may be underestimated here.
In addition, if cellulosic biomass, instead of pure glucose, can be used as the feedstock (as in the NREL process), then the process before fermentation includes biomass pre-treatment and enzymatic hydrolysis . The stream after hydrolysis contains 240 g/L sugar, and the cost of pre-treatment and hydrolysis is ~ 0.28 $/kg (pure sugar basis), i.e., a 53% feedstock cost saving compared to the 0.6 $/kg pure glucose price. We do not consider the use of cellulosic biomass in this study due to its limited applications in industry. Nonetheless, the readers can account for this technology by reducing the current glucose cost by 53%.
Note that this work is intended to be a high-level synthesis and analysis of bio-based terpene processes. Our goal is to identify the key cost drivers and provide target values for the researchers working in the area. If the key parameters discussed herein are substantially improved in the future, so that a positive profit margin can potentially be achieved, then more detailed studies accounting for aspects such as contamination prevention and microbial cell growth control would be required.
Finally, we note that the approaches discussed in this work can be used to study processes for the production of other extracellular, insoluble, and light (in terms of density compared to water) products, such as fatty acids.
This work focuses on the process synthesis, simulation, and techno-economic evaluation of microbial terpenes production. We first analyzed the vapor–liquid equilibrium, which explained the counterintuitive experimental phenomenon where terpenes such as limonene (normal boiling point 176 °C) are often found to be 100% present in the vapor phase after fermentation (at ~ 30 °C). Based on this analysis, we further proposed three alternative process configurations, demonstrated with three different case studies.
We estimated that the total unit production costs of the three cases are 465, 5.82, and 2.02 $/kg, respectively. We also identified the key cost drivers to be (1) feed glucose concentration, (2) yield, and (3) VVM. We further showed how these drivers impact costs and the selectin of the corresponding configurations. We found that the production of limonene, based on current literature experimental data, is economically infeasible and that higher glucose concentration and yield are key to lowering the cost.
Finally, we extended the assessment to account for different process parameters, terpene products, strategies to address terpene toxicity (microbial engineering vs. use of solvent), and cellulosic biomass as a feedstock. After studying the economics of 12 terpenes, limonene appears to be the most reasonable short-term target.
The framework and suite of methods presented herein are applicable to a wide range of extracellular insoluble chemicals with density lower than that of water, such as fatty acids. Therefore, the proposed methods can provide guidance and useful insights into the development of bio-based production systems for terpenes and other bioproducts.
WW performed the VLE analysis, process synthesis, techno-economic assessment, and completed the writing of the manuscript. CTM supervised the work, provided research directions and resources, and provided feedback on writing. Both authors read and approved the final manuscript.
We thank Profs Brian F. Pfleger and Jennifer L. Reed and Nestor J. Hernandez Lozada for fruitful discussions on terpene production and bio-conversions.
The authors declare that they have no competing interests.
Availability of data and materials
Data generated or analyzed during this study are included in this published article and its additional files. Additional information is available from the corresponding author on reasonable request.
Consent for publication
Ethics approval and consent to participate
This work was funded by National Science Foundation through the Emerging Frontiers in Research and Innovation program (EFRI-1240268), and the DOE Great Lakes Bioenergy Research Center (DOE BER Office of Science DE-SC0018409).
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