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Bio-Aromatics Production via Fermentation – A Case Study on p-

Hydroxybenzoic Acid (pHBA) - Process Modeling and Techno-Economic
Assessment (TEA) using SuperPro Designer.

Preprint · March 2021

DOI: 10.13140/RG.2.2.32011.21286

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 Bio-Aromatics via Fermentation

A Case Study on p-Hydroxybenzoic

Acid (pHBA) Production

Modeling and Cost Analysis with

SuperPro Designer®
by

Rafael da Gama, Nikiforos Misailidis and Demetri Petrides

This is the ReadMe file of a SuperPro Designer example that analyzes the production of Bio-Aromatics
via fermentation. It is a case study on the production of p-Hydroxybenzoic Acid (pHBA). The flowsheet of
the process is appended to the bottom of this document. You may test-drive the model by downloading
the functional evaluation edition of SuperPro Designer from the downloads page of our website
(www.intelligen.com). The files of this example can be found in the Examples \ Bio-Materials \
BioAromatics folder. The default installation path of the SuperPro Designer Examples folder follows
below.

C:\ Users \ Public \ Public Documents \ Intelligen \ SuperPro Designer \ v12 \ Process Library \ Examples

If you have any questions about this example and SuperPro Designer in general, please send an email
message to dpetrides@intelligen.com

INTELLIGEN, INC.
Simulation, Design, and Scheduling Tools
For the Process Manufacturing Industries
www.intelligen.com
Introduction

Aromatic compounds form an important class of organic molecules that find applications in a wide range of
fields, such as the manufacturing of polymers (plastics, fibers, coatings, resins etc.), pharmaceuticals,
foods, personal care products, cosmetics, and chemicals. Nowadays, aromatic compounds are almost
exclusively produced from fossil sources and, in particular, from benzene, toluene and xylene (BTX)
obtained from oil refining [1,2]. Nevertheless, living organisms are also capable of synthesizing aromatic
molecules, which opens the door to potentially greener alternatives to manufacture aromatic chemicals.
Plants, in particular, naturally produce a wide variety of aromatic molecules, including aromatic amino acids,
monolignols and a myriad of secondary metabolites, such as flavonoids, coumarins and stilbenoids [2,3].
Fungi and bacteria are also able to synthesize aromatics, including aromatic amino acids and secondary
metabolites. In both plants and microbes, the synthesis of aromatic compounds usually relies on the so-
called shikimate pathway (Figure 1). The shikimate pathway is a seven-step metabolic route that requires
two starting materials from central metabolism: phosphoenolpyruvate (PEP), from the EMP pathway; and
erythrose-4-phosphate (E4P), from the pentose-phosphate pathway. First, PEP and E4P are combined to
form 3-deoxy-D-arabinoheptulosonate-7-phosphate (DAHP), which is the key precursor of the shikimate
pathway. After three enzymatic reactions, DAHP is converted into shikimate, after which the pathway is
named. Shikimate is then transformed into chorismate via three additional steps. Chorismate may be
regarded as a key metabolic node in aromatic biosynthesis, given that the production of most aromatic
compounds goes through chorismate. In particular, all three aromatic amino acids – L-phenylalanine,
L-tryptophan and L-tyrosine – are ultimately synthesized from chorismate [1–6].

Figure 1: Schematic representation of the shikimate pathway. It starts with the condensation of two key precursors
from central metabolism, erythrose 4-phosphate and phosphoenolpyruvate. After seven reaction steps, chorismate is
produced. Most natural aromatic molecules are then derived from chorismate. DAHP: 3-deoxy-D-arabino-
heptulosonate-7-phosphate; EPSP: 5-enolpyruvylshikimate-3-phosphate. Source: [7].
p-Hydroxybenzoic Acid (pHBA)

p-hydroxybenzoic acid (pHBA), also known as 4-hydroxybenzoic acid (4HBA), is an aromatic compound
(Figure 2) that is employed in the synthesis of various products, such as dyes, pesticides, and plastics.
Esters of pHBA, commonly known as parabens, are also widely used as preservatives in foods, cosmetics
and pharmaceuticals, due to their antimicrobial activity and low toxicity [8]. As a monomer, pHBA can also
be used to produce liquid polymer crystals, which can be spun into fibers with exceptional chemical
resistance, thermal stability, and tensile properties. These fibers are used in the manufacturing of
automotive parts, electronics and even space exploration equipment [9,10].

Figure 2: Molecular structure of 4-hydroxybenzoic acid (pHBA).

pHBA is also found in Nature; in fact, bacteria such as E. coli synthesize pHBA as an intermediate to
produce ubiquinone, while plants synthesize phenylpropanoids that degrade into pHBA [2]. Currently,
however, the world production of pHBA – approximately 50,000 metric tons (MT) per year [11] – is entirely
met by oil-based chemical synthesis [12]. This synthesis usually follows the so-called Kolbe-Schmitt
process: in the first step, phenol is converted into potassium phenolate by the addition of potassium
hydroxide; then, in the second step, potassium phenolate is carboxylated under a high pressure of CO 2
(~0.45 MPa) and a high temperature (above 220 °C), which generates pHBA [8]. However, this method
suffers from relatively low yields and selectivity. For these reasons, and also for safety and environmental
concerns, the interest in producing pHBA biologically has increased over the last years [12,13]. Moreover,
for certain applications, such as food, cosmetic and personal care products, biological production is
advantageous from a marketing standpoint. In addition, the decarboxylation of microbially-produced pHBA
could offer a route to manufacture phenol from renewable sources [14].

pHBA biosynthesis

The scientific and patent literature of the last couple of decades describes several approaches to the
biological production of pHBA. In fact, the production of pHBA has been implemented in tobacco plants,
various bacteria (Escherichia coli, Klebsiella pneumonia, Corynebacterium glutamicum, Pseudomonas
putida) and baker’s yeast. Although these studies employ diverse metabolic engineering approaches, a
common strategy is to combine the shikimate pathway with the enzyme chorismate lyase, which converts
chorismate into pHBA in a single step (in E. coli, this enzyme is named UbiC and is normally involved in
ubiquinone synthesis) [5].

To this date, the best published results of pHBA biosynthesis have been achieved with metabolically
engineered bacteria, particularly E. coli (12 g/L) and C. glutamicum (36.6 g/L) [5]. With baker’s yeast, a
maximum titer of 2.9 g/L has been obtained. A key difference between the production of pHBA in yeasts
and bacteria, however, is that yeasts tolerate lower pH levels, which is particularly relevant in the context
of the production of organic acids such as pHBA. Consequently, yeast processes can be conducted without
continuously adding a mineral base for pH adjustment, as opposed to bacterial processes [9]. Other than
saving material and economic resources, this has implications on downstream processing, given that the
addition of base converts pHBA into its corresponding salt. It is also worth noting that pHBA, in contrast
with pHBA salts, is scarcely soluble in water; in fact, it saturates at approximately 5 g/L at 25 °C [15]. In
practice, this means that bacterial cultures end up generating (soluble) pHBA salts, which need to be
acidified if pHBA in acid (uncharged) form is desired. Yeast cultures without pH control, on the other hand,
will produce pHBA in acid form, and, if pHBA levels get sufficiently high, the molecule will precipitate inside
the bioreactor.

pHBA recovery and purification

Although works on microbial production of pHBA tend to focus on pHBA synthesis and to neglect product
recovery and purification, the recovery and purification of chemically-produced mono-hydroxybenzoic acids
such as pHBA and 2-hydroxybenzoic acid (a.k.a. salicylic acid) can provide some guidance to the process
design of microbial pHBA production. The chemical synthesis of mono-hydroxybenzoic acids through the
Kolbe-Schmitt method typically generates an alkali metal salt of the organic acid. In the case of salicylic
acid, for example, sodium salicylate is produced. Product recovery begins with dissolving the salicylate in
water; next, the solution is treated with activated carbon for decolorization [16,17]. After that, the solution is
acidified with a strong mineral acid to precipitate the product under its acid (uncharged) form, and the
resulting solid is finally recovered by a solid-liquid separation such as centrifugation [16].

The downstream processing of other microbially-produced organic acids such as citric acid, lactic acid or
succinic acid can also be useful to develop a recovery and purification scheme for microbial pHBA. Unit
operations typically employed to recover and purify microbial carboxylic acids include adsorption,
extraction, precipitation, crystallization, electrodialysis, evaporation and distillation [18]. Crystallization
appears to be a natural choice for pHBA, in view of its remarkably low solubility in water and propensity to
crystallize.
Process Description

The model of this example was developed in collaboration with Dr. Norbert Kohlheb and Dr. Jens Krömer
from the Helmholtz Institute at Leipzig, Germany, drawing from their extensive experience with microbial
production of aromatic organic acids, as well as peer-reviewed articles, patents, and our own engineering
judgement. We assume that pHBA is produced by an improved Corynebacterium glutamicum strain, grown
on sucrose and mineral medium. The bacterial culture is carried out aerobically, in fed-batch mode, for 36
hours. The temperature is controlled at 35 °C, and the pH level is maintained at 7.0, with the addition of
sodium hydroxide, so that pHBA is converted into its sodium salt (sodium p-hydroxybenzoate) as it is
synthesized during microbial culture. At the end of the bacterial culture, the broth is clarified by
centrifugation, and the supernatant is concentrated by reverse osmosis. After that, the concentrate is
subjected to activated carbon treatment for decolorization. Next, the solution of pHBA salt is acidified with
sulfuric acid, to convert the pHBA salt into pHBA (uncharged form), which is then crystallized in a separate
vessel. Finally, the pHBA crystals are separated from the mother liquor using basket centrifugation, and the
product is dried with the aid of a fluid-bed dryer.

The annual production rate was defined as 30,000 MT. This represents around 60% of the entire current
world production of pHBA; however, the demand for pHBA has been growing strongly, along with the
demand for similar aromatic organic acids, which could conceivably be manufactured in the same
production plant. Such production scale requires a broth volume of 257 m3 per batch, generated by eight
staggered fermentors of 321 m3 each, and a final output of 23 MT of dry pHBA per batch (approximately 4
MT/h).

For reporting and analysis purposes, the process has been divided into four sections:

• Media Preparation (black icons)

• Fermentation (teal icons)
• Primary Recovery (dark yellow icons)
• Purification (violet icons)
Flowsheet sections in SuperPro are simply sets of related unit procedures (processing steps). For
information on how to specify flowsheet sections and edit their properties, please consult Chapter 8.1 of
the SuperPro manual. The contents of each of this example’s flowsheet sections are described in greater
detail below. The flowsheet of the process is appended to the bottom of this document.
Media Preparation

The bacterial culture requires four major medium compounds: sucrose, which is the main carbon source;
sodium dihydrogenphosphate, which is a source of phosphorus; diammonium sulfate, which is a source of
nitrogen and sulfur; and ammonium chloride, which is another source of nitrogen. All these components
are dissolved in water, heat-sterilized and stored in a flat-bottom tank prior to use; sucrose is prepared as
a 50% w/w solution, while the three salts are prepared as a single solution containing 4.2 g/L of diammonium
sulfate, 44.8 g/L of sodium hydrogenphosphate, and 165.4 g/L of ammonium chloride. The same sterilizer
(PZ-101) is used by both sucrose and salt solutions (for more information on sharing equipment between
procedures, please refer to the Industrial Enzymes example in the Bio-Materials folder). Sodium hydroxide
is used in the process as well, for pH control; it is prepared as a 50% w/w solution and stored in a flat-
bottom tank too. Each storage tank then feeds its solution to the seed fermentors and main fermentors of
the process (as we will explain in the Fermentation Section, this process comprises two seed fermentation
steps and one main fermentation step), by means of a flow distribution procedure. In the case of sucrose,
the solution stream that feeds each fermentation procedure is further split into two streams, because each
fermentation step goes through a batch phase and a fed-batch phase. Sterile water is also added to the
fermentors to dilute the medium components to the appropriate concentrations to begin the batch phase of
the bacterial culture.

We assumed that the plant is built in Brazil, which is the largest producer of sugar in the world and has a
relatively low labor cost (the base salary of the plant operators was thus adjusted to reflect Brazilian wages).
For the cost of sucrose, the average May 2019 spot price of sugar was assumed (0.12 US$/lb or
0.26 US$/kg) [19]. For all other medium components and process chemicals, prices were sourced from US
import statistics [20].

Fermentation Section

The Fermentation Section encompasses the fermentation steps and the production of sterile air required
by the fermentors. We consider the use of gram-positive bacterium Corynebacterium glutamicum for the
synthesis of pHBA, since this microorganism has generated the best pHBA titers reported in the literature
to date. However, the reported yields, rates and titers for pHBA production in C. glutamicum are still sub-
optimal; consequently, we assumed the use of an improved strain of C. glutamicum that can achieve 90%
of the theoretical maximum carbon yield, as predicted by elementary flux mode analysis [21]. We also
assumed a productivity of 3.3 g /(gDW∙h) of pHBA. We believe this value is realistic, given that a productivity
of 4.0 g/(gDW∙h) has been demonstrated for lysine production with the same microorganism [22].
Considering that the shikimate pathway also provides the aromatic amino acids phenylalanine, tyrosine and
tryptophan, and that their combined anabolic demand exceeds the demand for lysine [23], it is reasonable
to assume that at least a comparable flux capacity exists for both the lysine and the shikimate pathways.
Regarding the configuration of the fermentation steps, we consider a sequence of two seed fermentation
procedures (P-22/SFR-201 and P-24/SFR-202) and one main fermentation procedure (P-26/FR-201), with
an expansion factor of 10x between SFR-201 and SFR-202, and of 10x between SFR-202 and the batch
(initial) volume of FR-201 as well. The first seed fermentation step is seeded with a suspension of 50 gDW/L
of biomass. The seed fermentation steps themselves reach 30 gDW/L of biomass. The fermentation
duration is equal to 12 h in the seed fermentation steps, and 36.16 h in the main fermentation step. In order
to start a new batch every 6 h, four staggered seed fermentors were assigned to the first seed fermentation
step; four staggered fermentors were assigned to the second seed fermentation step; and eight staggered
fermentors were assigned to the main fermentation step (for details on the use of staggered equipment,
please refer to the Farnesene example in the Bio-Materials folder). Each fermentation step starts with a
batch phase and, when the sugar is almost depleted, a fed-batch phase begins by adding supplementary
sugar (sucrose 50% w/w). The bacterial cultures are conducted at 35 °C, with an aeration rate of 0.5 VVM
(volume of air per volume of liquid per minute). This air stream is generated by passing air through an air
filter and then through a compressor, to increase its pressure by 2 bar; after that, the air stream is distributed
to the fermentors using a flow-distribution procedure. The use of a flow-distribution procedure, rather than
a flow-splitting procedure, allows SuperPro to back-calculate the required volume of air input to satisfy the
aeration demand of the fermentors, according to the value specified earlier (0.5 VVM). The flow-distribution
procedure is further explained in the Farnesene example.

The microbial growth in each seed fermentation step is represented by the following mass stoichiometry.

0.29 (NH4)2SO4 + 11.42 NH4Cl + 3.10 NaH2PO4 + 100.00 Sucrose → 45.01 Biomass + 69.80 CO2

Microbial growth also happens in the main fermentation step; in addition, pHBA is synthesized at this point.
The following mass stoichiometry was employed to represent this step:

0.12 (NH4)2SO4 + 4.87 NH4Cl + 1.32 NaH2PO4 + 100.00 Sucrose → 11.22 Biomass + 55.91 CO2 + 39.18
pHBA (aq)

It is worth noting that the solubility of pHBA in pure water is quite low, only 5.0 g/L at 25 °C. However, as
mentioned earlier, the pH of the fermentation is controlled at 7.0 and, for that reason, an alkali base (sodium
hydroxide) is added in all fermentation steps. Consequently, the pHBA produced by the microorganism in
the main fermentation step is entirely converted into its sodium salt, sodium p-hydroxybenzoate, that has a
much higher solubility. In fact, this is likely the reason why titers upwards of 60 g/L pHBA have been reported
in the literature (10). The neutralization of pHBA by sodium hydroxide is represented by the following molar
stoichiometry:

1 NaOH + 1 pHBA (aq) → 1 sodium p-hydroxybenzoate + 1 H2O

We assume that a final titer of 102 g/L of pHBA salt (equivalent to 88 g/L of pHBA) could be achieved. The
total broth volume generated by the production fermentors is 257 m3 per batch. Each staggered fermentor
used in the production step has a vessel volume of 321 m3; each staggered fermentor used in the first seed
fermentation step has a vessel volume of 1.7 m3; and each staggered fermentor used in the second
fermentation step has a vessel volume of 17 m3.

At the end of the fermentation process, the entire broth is transferred to a surge tank (P-28/HT-201) which
functions as a buffer storage between the fermentation section that operates in batch mode and the
downstream sections which run mostly in continuous mode.

Primary Recovery Section

In this section, the broth is clarified by centrifugation and concentrated by reverse osmosis. The
fermentation broth is first sent to a disk-stack centrifuge (P-29/DS-301), which removes 97% of the cell
mass. The heavy stream of this centrifuge is diluted with water and centrifuged again in a second centrifuge
(P-31/DS-302) to minimize pHBA losses; the supernatants of both centrifuges are combined and sent to a
dead-end microfilter (P-38/DE-301) that removes any residual biomass present in the mixture. Next, the
solution containing pHBA salt is concentrated to 250 g/L by reverse osmosis (P-39/RO-301), and the
concentrate is sent to the Purification Section.

To minimize waste treatment costs and the environmental footprint of the process, the solids stream coming
out of the second centrifuge is concentrated in a triple-effect evaporator (P-33/EV-301) and dried with hot
air in a rotary dryer (P-34/RDR-301). The resulting dry material has a high concentration of biomass and
may be sold as fertilizer. The hot air utilized by the dryer is produced by burning natural gas.

Purification Section

In this section, the pHBA salt is decolorized, converted into pHBA proper, crystallized, and dried. First, the
solution containing the pHBA salt passes through an activated carbon column (P-40/GAC-401) for
decolorization; this step primarily removes organic impurities. Next, the solution is acidified with sulfuric
acid in a continuous neutralization vessel (P-41/AT-401). Within the NEUTRALIZE operation of that
procedure, in the Oper.Cond’s tab, an acid excess of 5% was specified. In the Reaction tab, the following
equation was defined:

2 pHBA Salt (aq) + 1 H2SO4 (aq) → 2 pHBA (aq) + 1 Na2SO4

where the coefficients are molar. The low solubility of the protonated form of pHBA (5 g/L at 20 °C) is then
exploited by crystallizing the acid in a continuous stirred-tank reactor (P-42/CR-401). Within the REACT
operation of that procedure, in the Oper.Cond’s tab, the temperature was set to 5 °C, to maximize the
percentage of pHBA crystallized; in the Reactions tab of the same procedure, a simple phase-change
equation was introduced:

1 pHBA (aq) + 1 H2O → 1 pHBA*H2O (solid)

Notice that pHBA crystallizes in the form of a monohydrate. The reaction conversion was set to achieve a
final concentration of pHBA (aq) in solution equal to 5 g/L.

The pHBA crystals are separated from the mother liquor with the aid of five parallel basket centrifuges (P-
43/BC-401). Within the FILTER operation of that procedure, in the Oper.Cond’s tab, the Particulate
Component Retention was set to 98% for pHBA*H2O and 0 for the other process components. In addition,
the cake loss on drying (LOD) was specified as 8% w/w. A WASH operation was also added to that
procedure to wash the cake formed by the previous operation. In the Oper.Cond’s tab of the WASH
operation, the amount of the Wash-In stream was defined as 2.0 volumes per volume of cake. Finally, the
cake, containing approximately 92.5% of pHBA*H2O, is dried using a fluid-bed dryer (P-45/FBD-401). In
this step, the pHBA crystals are dried with hot air until most of the water is removed. To model that, in the
Oper.Cond’s tab of the DRY operation, the option “Calculated Based on Final LOD” was selected, “water”
was specified as a volatile component and a “Final LOD” of 0.5% was defined. Those specifications lead
to a product that is 99.5% pure. As in the drying of biomass described earlier, the hot air used to dry the
pHBA crystals is generated by burning natural gas.

Process Scheduling and Debottlenecking

The overall batch time for this process is approximately 85.4 h (3.6 days). This is the time elapsed from the
start of a given batch (i.e., the preparation of media, sodium hydroxide, etc.) to the end of that batch (the
generation of pure product). However, the batch cycle time is only 6 hours because the individual
procedures in this process are much shorter than the overall batch time, and multiple (staggered) equipment
items are used in some parts of the process.

To visualize the process schedule, click Charts Equipment Occupancy Multiple Batches. This will
generate the Equipment Occupancy Chart (EOC). A portion of the EOC, showing 16 consecutive batches
of this process, is displayed in Error! Reference source not found.. The upper portion of the chart shows
that eight production fermentors are employed in this process: FR-201, FR-201b, FR-201c, FR-201d, FR-
201e, FR-201f, FR-201g and FR-201h. In addition, it shows that they operate in Stagger Mode (out of phase
with each other). Likewise, a set of four staggered seed fermentors carry out the first seed fermentation
step (SFR-201, SFR-201b, SFR-201c, SFR-201d), and another set of four staggered seed fermentors
(SFR-202, SFR-202b, SFR-202c, SFR-202d) carry out the second seed fermentation step. This
configuration enables the plant to initiate a new batch every 6 h, even though each fermentation procedure
takes much longer. Further details on specifying equipment in Stagger Mode can be found in the Farnesene
example, in the Bio-Materials folder.

The bottom portion of the chart displays downstream equipment. It has a blockish aspect, without gaps
between the bars; this indicates that the downstream procedures operate in a seemingly continuous
manner. In fact, most downstream procedures run in continuous mode; however, a few of them, such as
reverse osmosis and activated carbon adsorption, run in batch mode, though they do so with multiple cycles
per batch. Given that each cycle of these procedures was specified to tightly follow the previous one, they
appear to be continuous. For more information on using continuous procedures in batch flowsheets, or
procedures that cycle multiple times per batch, please refer to the Industrial Enzymes example in the Bio-
Materials folder.

Another view of the process schedule is provided by the Operations Gantt chart (in the MS Project style).
This chart displays detailed scheduling information for one or multiple batches. The Gantt chart for a single
batch is generated by selecting ChartsGantt ChartsOperations GC. Figure 4Error! Reference source
not found. displays a portion of that Gantt chart, illustrating the scheduling of operations in the seed and
production fermentors. The dark blue and cyan bars represent the durations of procedures and operations,
respectively.

The Gantt chart enables users to visualize the execution of a batch process in detail. It also facilitates
editing of batch recipes. Double-clicking on any of its bars brings up the dialog of the corresponding entity
(e.g., operation, procedure, recipe, etc.). The simulation calculations can then be redone, and the chart can
be updated by clicking on the refresh button of the chart ( ).

Furthermore, SuperPro can export its scheduling data to MS Project by selecting FileExport to MS
Project XML File. Likewise, SuperPro can export its recipe data to SchedulePro by selecting FileExport
to SchedulePro’s Recipe DB. SchedulePro is a resource management, production planning and
scheduling tool available from Intelligen. Please consult the SuperPro manual or its Help facility for
information on these two exporting options.
 CIP

Media Preparation

Fermentation

Primary Recovery

Purification

Figure 3: Equipment Occupancy Chart (EOC) for 16 consecutive batches, showing the equipment used in each section.
Figure 4: Portion of the Operations Gantt chart for a single batch of pHBA production.
Material Requirements

Table 1 displays the raw material requirements in kg/yr, kg/batch, and kg/kg of MP for this process (“MP”
stands for main product, which is pHBA in this case). This table was extracted from the RTF version of the
Materials & Streams report, which can be generated by selecting Reports Materials & Streams from
the main menu bar of SuperPro. The format of the report can be specified through the dialog that is
displayed when you select Reports Options from the main menu bar of SuperPro. As expected, Table
1 shows the carbon source (sucrose) is by far the most significant raw material in the process in terms of
mass (apart from air and water).

Table 1: Material Requirements for the Entire Process.

BULK MATERIALS (Entire Process)

Material kg/yr kg/batch kg/kg MP
Air 830,734,733 636,090.91 27,691.16
Biomass 8,034 6.15 0.27
H2SO4 (98% w/w) 10,594,739 8,112.36 353.16
H3PO4 (2%) 13,628,987 10,435.67 454.30
NaOH (0.5 M) 29,956,924 22,937.92 998.56
Natural Gas 4,053,979 3,104.12 135.13
Salts 5,210,698 3,989.81 173.69
Sodium Hydroxide 8,566,894 6,559.64 285.56
Sucrose 78,120,748 59,816.81 2,604.02
Water 564,791,639 432,459.14 18,826.39
TOTAL 1,545,667,375 1,183,512.54 51,522.25

Cost Analysis

SuperPro Designer performs thorough cost analysis, estimating capital (CAPEX) as well as operating
(OPEX) costs, and generates the following three pertinent reports (through the Reports menu): the
Economic Evaluation Report (EER), the Cash Flow Analysis Report (CFR), and the Itemized Cost Report
(ICR).

Table 2 displays the Executive Summary of the Economic Evaluation Report. The total capital investment
was estimated in approximately $118 million and the unit production cost in 1.98 $/kg of product. Assuming
a selling price of 2.54 $/kg of pHBA, the gross margin would be 22.6%; the return on investment, 20.6%;
and the payback period, 4.9 years. These metrics suggest that the proposed process would be a reasonable
investment.
Table 2: Executive Summary

EXECUTIVE SUMMARY (2019 prices)

Total Capital Investment 117,798,000 $
Capital Investment Charged to This Project 117,798,000 $
Operating Cost 59,380,000 $/yr
Main Revenue 76,200,000 $/yr
Other Revenues 528,657 $/yr
Total Revenues 76,729,000 $/yr
Batch Size 22.97 MT MP
Cost Basis Annual Rate 30,000 MT MP/yr
Unit Production Cost 1,979.32 $/MT MP
Unit Production Revenue 2,557.62 $/MT MP
Gross Margin 22.61 %
Return On Investment 20.57 %
Payback Time 4.86 years
IRR (After Taxes) 17.61 %
NPV (at 7,0% Interest) 123,821,000 $
MP = Total Flow of Stream 'Dry pHBA'

Figure 5 displays the annual operating cost breakdown, which is also part of the EER. This type of chart
can be included in the report by selecting Reports Options and activating the Include Charts option on
the lower right corner of the dialog. In the current example, this chart shows that the facility dependent cost,
raw materials and utilities contribute the most to the operating cost, accounting for 35%, 26% and 22%,
respectively.

Table 3 (also extracted from the EER) indicates that the raw material cost is mostly due to sucrose, which
is the main carbon source and accounts for 55.4% of the total cost of raw materials. Acids and bases
account for most of the remaining raw material costs. The unit cost of each raw material can be viewed in
the program by clicking on the Process Explorer button in the main toolbar, or by selecting View Process
Explorer on the main menu bar of SuperPro.
Figure 5: Annual Operating Cost Breakdown

The large share of the facility-dependent cost in the OPEX suggests that a potential strategy to make this
process more economically attractive would be the reduction of the total equipment purchase cost, possibly
through process intensification. For instance, in situ product crystallization might be an alternative to
improve the fermentation process while performing the primary recovery of the product at the same time.
As such, the downstream process could be performed in fewer steps, and the total equipment purchase
cost might decrease.

In any case, many assumptions underlie this kind of analysis, such as the selling price of pHBA, the costs
of raw materials, the actual fermentation yields and purification efficiency, etc. As a result, the actual
economics of such a plant may be substantially better or worse than the current projection. Indeed, a useful
exercise is to perform “what-if” analyses with SuperPro to determine the impact of various changes (such
as a change in the yield of pHBA on sucrose). This allows one to understand the potential risks and rewards
of a project under different sets of assumptions. What-if scenarios can be evaluated individually (by simply
changing parameter values manually and re-running the simulation to see the results), or they can be
automated through MS Excel. For information on how to drive SuperPro through MS Excel, please consult
the examples in the “…EXAMPLES \ COM” folder. Related information is available in the Help Facility of
the tool, which can be accessed by selecting Help  COM Interface and Library.
Table 3: Cost of Raw Materials.

MATERIALS COST - PROCESS SUMMARY

Unit Cost Annual Annual Cost
Bulk Material %
($) Amount ($)
Air 0.00 830,734,733 kg 0 0.00
Biomass 0.00 8,034 kg 0 0.00
H2SO4 (98% w/w) 0.08 10,594,739 kg 808,696 5.23
H3PO4 (2%) 0.16 13,628,987 kg 2,175,050 14.07
NaOH (0.5 M) 0.01 29,956,924 kg 212,365 1.37
Natural Gas 0.50 15,258 m3(STP) 7,629 0.05
Salts 0.14 5,210,698 kg 748,626 4.84
Sodium Hydroxide 0.32 8,566,894 kg 2,715,705 17.57
Sucrose 0.11 78,120,748 kg 8,564,299 55.41
Water 0.40 564,792 MT 224,222 1.45
TOTAL 15,456,593 100.00

NOTE: Bulk material consumption amount includes material used as:

- Raw Material
- Cleaning Agent
- Heat Transfer Agent (if utilities are included in the operating cost)
References

[1] Lee JH, Wendisch VF. Biotechnological production of aromatic compounds of the extended
shikimate pathway from renewable biomass. J Biotechnol 2017;257:211–21.
doi:10.1016/j.jbiotec.2016.11.016.

[2] Huccetogullari D, Luo ZW, Lee SY. Metabolic engineering of microorganisms for production of
aromatic compounds. Microb Cell Fact 2019;18:1–29. doi:10.1186/s12934-019-1090-4.

[3] Wu F, Cao P, Song G, Chen W, Wang Q. Expanding the repertoire of aromatic chemicals by
microbial production. J Chem Technol Biotechnol 2018;93:2804–16. doi:10.1002/jctb.5690.

[4] Noda S, Kondo A. Recent Advances in Microbial Production of Aromatic Chemicals and Derivatives.
Trends Biotechnol 2017;35:785–96. doi:10.1016/j.tibtech.2017.05.006.

[5] Averesch NJH, Krömer JO. Metabolic engineering of the shikimate pathway for production of
aromatics and derived compounds-Present and future strain construction strategies. Front Bioeng
Biotechnol 2018;6. doi:10.3389/fbioe.2018.00032.

[6] Wang J, Shen X, Rey J, Yuan Q, Yan Y. Recent advances in microbial production of aromatic natural
products and their derivatives. Appl Microbiol Biotechnol 2018;102:47–61. doi:10.1007/s00253-017-
8599-4.

[7] Zucko J, Dunlap WC, Shick JM, Cullum J, Cercelet F, Amin B, et al. Global genome analysis of the
shikimic acid pathway reveals greater gene loss in host-associated than in free-living bacteria. BMC
Genomics 2010;11:628. doi:10.1186/1471-2164-11-628.

[8] Ritzer E, Sundermann R. Hydroxycarboxylic Acids, Aromatic. In: Elvers B, editor. Ullmann’s Fine
Chem. 1st ed., Weinheim, Germany: WILEY‐VCH Verlag; 2014, p. 715–23.

[9] Averesch NJH, Prima A, Krömer JO. Enhanced production of para-hydroxybenzoic acid by
genetically engineered Saccharomyces cerevisiae. Bioprocess Biosyst Eng 2017;40:1283–9.
doi:10.1007/s00449-017-1785-z.

[10] Choi WJ, Byun JW, Ahn JH, Ha YW, Seo JH. Process of biologically producing p-hydroxybenzoic
acid. EP 2 698 435 A1, 2013.

[11] Krömer JO, Ferreira RG, Petrides D, Kohlheb N. Economic Process Evaluation and Environmental
Life-Cycle Assessment of Bio-Aromatics Production. Front Bioeng Biotechnol 2020;8.
doi:10.3389/fbioe.2020.00403.

[12] Wang S, Bilal M, Hu H, Wang W, Zhang X. 4-Hydroxybenzoic acid—a versatile platform

 intermediate for value-added compounds. Appl Microbiol Biotechnol 2018;102:3561–71.
doi:10.1007/s00253-018-8815-x.

[13] Yu S, Plan MR, Winter G, Krömer JO. Metabolic engineering of Pseudomonas putida KT2440 for
the production of para-hydroxy benzoic acid. Front Bioeng Biotechnol 2016;4:1–10.
doi:10.3389/fbioe.2016.00090.

[14] Kitade Y, Hashimoto R, Suda M, Hiraga K, Inui M. Production of 4-hydroxybenzoic acid by an

aerobic growth-arrested bioprocess using metabolically engineered Corynebacterium glutamicum.
Appl Environ Microbiol 2018;84. doi:10.1128/AEM.02587-17.

[15] National Center for Biotechnology Information. PubChem Database. 4-Hydroxybenzoic acid,
CID=135 n.d. https://pubchem.ncbi.nlm.nih.gov/compound/4-Hydroxybenzoic-acid (accessed
January 21, 2020).

[16] Jansen G, Wolff P. Production of sodium salicylate. EP 0072095, 1981.

[17] Lindsey AS, Jeskey H. The Kolbe-Schmitt Reaction. Chem Rev 1957;57:583–620.
doi:10.1021/cr50016a001.

[18] López-Garzón CS, Straathof AJJ. Recovery of carboxylic acids produced by fermentation.
Biotechnol Adv 2014;32:873–904. doi:10.1016/j.biotechadv.2014.04.002.

[19] International Sugar Organization. International Sugar Organization - Daily Sugar Prices n.d.
https://www.isosugar.org/prices.php (accessed October 31, 2019).

[20] US International Trade Commission. US International Trade Commission Dataweb n.d.

https://dataweb.usitc.gov/trade/search/ (accessed November 18, 2020).

[21] Krömer JO, Nunez-Bernal D, Averesch NJH, Hampe J, Varela J, Varela C. Production of aromatics
in Saccharomyces cerevisiae-A feasibility study. J Biotechnol 2013.
doi:10.1016/j.jbiotec.2012.04.014.

[22] Becker J, Zelder O, Häfner S, Schröder H, Wittmann C. From zero to hero-Design-based systems
metabolic engineering of Corynebacterium glutamicum for l-lysine production. Metab Eng 2011.
doi:10.1016/j.ymben.2011.01.003.

[23] Stephanopoulos GN, Aristidou A a, Nielsen J. Metabolic Engineering: Principles and Methodologies.
1998.
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