Production of R-3-hydroxybutyrate (R-3-HB) from

Genetically Modified Escherichia Coli in a Medium with

Glucose
Maria A. Caicedo Garcia, Carlos Ariel Cardona.
Instituto de Biotecnologa y Agroindustria, Departamento de Ingeniera Qumica, Universidad Nacional de
Colombia sede Manizales, Cra 27 No. 64-60 , Manizales, Colombia

ABSTRACT
________________________________________________________________________________________________________
In this paper a techno-economic and environmental analysis for production of Poly-3-
hydroxybutyrate (PHB) for obtaining R-3-hydroxybutyrate (R-3-HB) from genetically modified
Escherichia Coli by fermentation of glucose. This scenario is going to simuled using the Aspen Plus
Software (Aspen Technologies Inc., USA) and experimental yields. The process simulation is a useful
tool to get an idea of the total production costs in a process including raw materials, energy
consumption and the installation costs to start the production of a desired product. We will do
sensibility analysis with the evaluation of the main variables of the process such as temperature,
concentration, pression, conditions of the fermentation with the propose of the optimize production
R-3-HB. In addition, life cycle inventories of energy consumption and carbon dioxide emissions from
the fermentation have been calculated for the copolyester production to examine the basic
environmental impact of the production, it is calculated with the waste algorithm reduction (WAR).
________________________________________________________________________________

Keywords
Bioproduction PHB, genetically modified Escherichia Coli, R-3-hydroxybutyrate (R-3-HB), Glucose,
Process simulation, renewable carbon source , life cycle inventory, CO2 emissions.

1. Introduction
Polyhydroxyalkanoates (PHAs) are natural, completely biodegradable, intracellular and can be
biocompatible polyesters synthesized by various bacterial species from renewable sources trough a
fermentation process under restricted growth conditions for nitrogen, phosphorus, sulfur and/ or
oxygen.[1] That are accumulated in a wide variety of microorganisms as carbon, energy stores and
reducing equivalents when carbon is in excess and other essential nutrients for cell growth are
limited. PHAs can be classified by the length of their carbon chain monomers: short-chain length (SCL)
PHA monomers containing C35, and medium-chain length PHA (MCL) monomers containing C6
14.[2] Among the (R)-hydroxyalkanoic acids, (R)-3-hydroxybutyric acid [(R)-3-HB] is considered
important and it is used in industrial and biomedical applications. Studies have shown that (R)-3-HB
has antimicrobial, insecticidal, and antiviral activities; and further it is very important to synthesis of
chemicals including antibiotics, vitamins, and pheromones [3]

The polyhydroxyalkanoates (PHA) are of interest because their material properties make them a
potential alternative to some petroleum-based thermoplastics. In many Studies it have been isolated
more than 240 bacterial strains and it have been found about 100 distinct structural variations. The
development in genetic engineering techniques have made it possible to express the PHA
biosynthetic genes in foreign hosts. Some examples of hosts includes such prokaryotic and eukaryotic
organisms as Escherichia coli ( Slater et al., 1988; Schubert et al., 1988 ; Peoples and Sinskey,
1989), Arabidopsis thaliana ( Poirier et al., 1992), , tobacco (Nakashita et al., 1999), Saccharomyces
cerevisiae ( Leaf et al., 1996; Poirier et al., 2001; Carlson et al., 2002 ; Breuer et al., 2002), Pichia
pastoris ( Poirier et al., 2002 ; Vijayasanakaran et al., 2005), Arxula adeninivorans ( Terentiev et al.,
2004), and insect culture cells (Williams et al., 1996). This polymer can be synthesized probably in
any cell environment but the enzymes should be present and that precursors , cofactors too in
appropriate concentrations for the synthesis reaction to occur. [4]
The inclusion of PHB as a new product is promising because this biopolymer is produced mainly from
sugars. In this case glucose can be used for PHB synthesis as the carbon source. However, the
selection of the PHB as a product in the Colombian context is based on the necessity to include
biopolymers in the market due to its increasing consumption and a fast increment on oil prices.[5]
The production cost of PHB depends on the yield and productivity of the microorganism, the
differents conditions as temperature, aeration, pH-value and stuff, the substrates for example the
carbon and nitrogen sources and the recovery and purification processes. The carbon source could
account for 2545% of the total production costs. In fact is very important and necessary to find
cheaper carbon sources as agroindustrial wastes can be good options and candidates because they
have some desired characterists and properties like low prices and simple accessible, further when
the agroindustrial wastes are used in differents processes or activities as substrates for the
production of PHB, the contribution of environmental is better because the final disposal is avoided
and this trouble is fewer. A variety of substrates such as glycerol (obtained from biodiesel
production), lignocelluloses material like glucose, whey and others have been used with several
microorganisms to obtain better yields and production of PHBs.[1]

Finding less expensive substrates would, therefore, be important for the commercialization of this
product. Many companies worldwide are developing products for PHAs. The current production of
P(3HB) is about 1000 t per annum[6]. A range of PHAs with 024% hydroxyvalerate has been
produced under the trade name of BIOPOL by Zeneca Bio Product and other manufacturers and sold
in the USA (under the trade name of PHBV), Germany and Japan. Table 1 showed manufacturer,
microorganisms and raw materials used for the production of biodegradable plastics.[7]
Table 1. Production of PHA by various bacteria. (Lee SY. 1996)

Abbreviations: P(3HB), poly(3-hydroxybutate; P(3HB-co-3HV), poly(3-hydroxybutyrate-co-3hydroxyvalerate);

P(3HV), poly(3-hydroxyvelerate); P(3hhx-co-3HO), poly(3-hydroxyhexanoate-co-3-hydroxyoctanoate).

In the present study, we propose a simple and efficient strategy to produce (R)-3-HB by
depolymerization of PHB with genetically modified E.Coli using glucose as substrate. Through the
simulation with Aspen Plus Software (Aspen Technologies Inc., USA) the main goal is tecno-
economical and environmental evaluate of the PHB production with the appropriate conditions to
obtain high yields of the desired product with the sensitivity analysis.

2. General framework
The PHB production process begins with the entry of raw materials (substrates and biomass), which
are converted into product by aerobic fermentation. The product obtained is intracellular (i.e. it
grows within the biomass) and crude (i.e., with an undesirable component composition in the broth),
such that a cell disruption and subsequent separation and purification steps are required (Dunn et
al., 2008). Consequently, the methodology for the process analysis has been divided into two parts:
(i) analysis of the fermentation reactor, where it is necessary to define the operating conditions to
achieve a high product yield and productivity; and (ii) analysis of PHB production process at the
industrial level through technical, economic and environmental assessments of a conceptual design,
so that the production plant is feasible. Fig. 1 shows, the general framework applied. Below is a brief
description of the methodology and the technical, economic and environmental criteria used for
decision-making in the initial stage of designing this process.[8]

Fig 1. General framework for technical-economic- environmental assessment of the production process design.[8]

3. Methodology
The methodology used to obtain R-3-HB is through of the Aspen plus Software simulation (Aspen
Technologies Inc., USA) with the knowledge-based strategy that considers both heuristic rules and
researchers experience. This tool allow to study the effect of the principal operation variables such
as reflux ratio, feed temperature, number of stages, conditions of fermentation, among others with
the sequential design procedure including a sensitivity analysis.

Escherichia Coli is going to simulate as a solid compound while the enzymes were simulated as non-
conventional compounds. For the thermodynamic analysis can be used the UNIFAC model.[9] The
fermentation process will be simulate based on a yielding approach where glucose is consumed in
fermentation stage where it occurs the mass cell growth and the PHB accumulation.

The simulation will be include three process stages : raw material purification, glucose fermentation
to PHB and the precursor R-3-HB and finally downstream processing with the purification and
recovery of desired product (R-3-HB).

The economic analysis is going to do with the Aspen Economic Analyzer Software (Aspen Technology,
Inc., USA). This package allow to estimate and know the capital cost of process units such as the
operating cost, besides the valuable data with the information supply by Aspen Plus and the data
consigned by the person that use this platform for specify conditions such as project location, Price
raw material among others. This analysis can be estimated in US dollars for a 10-year period at an
annual interest rate of 16%, considering the straight-line depreciation method and a 33% income tax.

It is very important to know the labor cost used to the raw material to obtain glucose. The operatives
and supervisors, the prices for electricity, water and pressure vapor used in the process, this values
can be based on Latin American conditions.
Calculation of energy consumption will be based on the thermal energy required by heat exchangers,
reboilers, flash drier units and the power supply required by the pumps.

Process simulation can be initially conducted using the commercial process simulator Aspen Plus
(Aspen Technologies Inc., USA). After mass and energy balances, analysis of the environmental impact
can carry out using the waste algorithm reduction (WAR) developed by the National Risk
Management Research Laboratory from the U.S. Environmental Protection Agency (EPA), which
evaluates the environmental friendliness of each process. This method proposes to add a reaction of
conservation over the potential environmental impact (PEI) based on the impact of input and output
flow rates from the process. For this application the EPA developed the software named WAR GUI.
The PEI for a given mass or energy quantity could be defined as the effect that those (energy and
mass) would have on the environment if they were arbitrarily discharged. The environmental impact
is a quantity that cannot be directly measured, however, it can be calculated from different
measurable indicators (Cabezas et al., 1999; Young and Cabezas, 1999). The WAR GUI software
incorporates the Waste Reduction Algorithm in process design measuring the following eight
categories: human toxicity by ingestion (HTPI), human toxicity by dermal exposition or inhalation
(HTPE), terrestrial toxicity potential (TTP), aquatic toxicity potential (ATP), global warming (GWP),
ozone depletion potential (ODP), photochemical oxidation potential (PCOP), and acidification
potential (AP).[10] It is very important to clarify that this environmental assessment only corresponds
to the possible impact generated in the productive process stage. In other words, the limits of the
system correspond to the processing facility. In this way a LCA is not considered, and a simpler
evaluation is carried out considering the mass and heat balances from the technical evaluation. The
latter also serve as a basis to compare different processing configurations.[5]

A sensitivity analysis is then carried out for many reasons, such as the need to determine which
parameters require further experimental research to improve process understanding and what
consequence results from the change of a certain input parameter.

The sensitivity analysis will obtain by increasing each parameter of the model with a given
percentage, leaving all others parameters constant and quantifying the change in the model outputs.
Thus, in a new study, these parameters should be studied and estimated experimentally with more
accuracy to ensure a satisfactory model prediction. In fact, the parameters can be directly related to
product and biomass yields, which depend strongly on the fermentation microorganism and the
operating conditions.

Another important environmental parameter is the greenhouse gas (GHG) emissions associated with
the chemical process. This evaluation corresponds to the potential emissions related to the
productive process stage of the supply chain. This only relates emissions associated with energy
balance of the processing stage.[10]

4. Results and discussion

4.1 Process Description

The process simulation allow to get an idea about of the production cost in this process including
differents aspects of it as raw materials, installation costs, energy consumption and also the
environmental conditions to choose the best option with the sensitivity analysis to obtain the desired
product and the process can be economically viable and environmentally friendly.
The purification of the raw material is very important in this process to get the glucose necessary for
the genetically modified Escherichia Coli.

Escherichia Coli will be simulated as a solid compound while the enzymes were simulated as non-
conventional compounds. For the thermodynamic analysis the UNIFAC model can be used. [9] The
enzymatic digestion is going to simulate based on a stoichiometric approach. Calculation of energy
consumption is going to base on the thermal energy required by heat exchangers, reboilers, flash
drier units and the power supply required by the pumps.

4.2 Fermentation process

Table 2 shows the PHB production, substrate consumption and biomass concentration from the
fermentation process using glucose as substrates. The fermentation step is carried out in a single
stage fermenter. In this level, cell growth and PHB accumulation occurs.[9]

Table 2 . PHB concentration, substrate consumption and biomass production in the fermentation step.

Feature Glucose
Initial substrate concentration (g/l) 20
Final substrate concentration [g/l) 10.7
Conversin 0.5
Concentration of PHB output (g/l) 4.8
Concentration of biomass output (g/l) 7.1
Fermentation time (h) 42

4.3 Separation and purification

The PHB separation and purification processes from a fermentation broth can be divided in three
steps: pretreatment, extraction, and purification. This process can start with a heat pretreatment
stage at 85 C, followed by a simultaneous chemical-enzymatic digestion. Then the disrupted cell
mass is discarded by a centrifugation process. The suspended PHB stream is then washed with a
H2O2 diluted stream (1.2 v/v%). Finally, the PHB is purified up to 99.9% weight by evaporation and
spray drying.[9]

The PHB production processes will be simulated using Aspen Plus (Aspen Technologies Inc., USA).
Thus, the design of the distillation columns in all cases can require the definition of the preliminary
specifications using the DSTWU shortcut method included in Aspen Plus (Aspen Technologies Inc.,
USA). This method uses the WinnUnderwoodGilliland procedure providing an initial estimate of
the minimum number of theoretical stages, the minimum reflux ratio, the localization of the feed
stage, and the products split. To perform the rigorous calculation of the distillation columns, the Rad-
Frac module (based on the MESH equations) can be used.[9]

4.4 Economic analysis

The PHB total production costs can be among 3.9 US$/kg and 3.3 US$/kg when glucose is used as
substrate. In general terms the lower PHB production costs can be to obtain when there is no solvent
recuperation in the separation scheme.[9]

4.5 Environmental analysis

To estimate accumulative energy and CO2 emissions using glucose like substrate could be used the
waste algorithm reduction (WAR), which evaluates the environmental friendliness of each process.

Figure 2 shows a variety of annual data from the United States Department of Agriculture (USDA) and
Department of Energy (DOE) for glucose. Based in literature a value of 8.1 MJ/kg-glucose was
provided for the cumulative energy use together with 2.4 MJ/kg for corn grain production quoted
from Shapouri et al. [16] and 4.4 MJ/kg for corn-wet milling to produce glucose. Therefore, we also
calculated cumulative energy use of glucose following the Shapouri's and Gerngross's procedures and
further expanded the calculations to estimate CO2 emissions. As in illustrated in Fig. 2, a value of 7.5
MJ/kg-glucose was obtained for the cumulative energy use together with 2.5 MJ/kg for corn grain
production and 4.9 MJ/kg [17,18] for corn wet milling to produce glucose. In addition, a value of 0.97
kg/kg-glucose was obtained for the cumulative CO2 emissions together with 0.15 kg/kg for corn grain
production and 0.35 kg/kg for corn-wet milling to produce glucose, taking into account of CO2 of 1.47
kg/kg-glucose absorbed or fixed from the air when corn plant has been grown up. The value of CO2
absorption was obtained from the MW 180 and carbon number 6 of glucose.[11]

Fig 2. Cumulative energy use and CO2 emissions of glucose before the fermentation stage

5. Conclusions
The microbiological conversion of glucose obtains of agroindustrial waste into PHB is an interesting
alternative to obtain added value products using this by product. Using simulation programs, the PHB
total production costs can be estimated at an industrial scale with substrate (glucose). Many aspects
of the process are very important and need attention to improve the economic viability and
sustainability of it, such as product technology, methods for separation and purification, process
engineering or the fermentation reactor, among others.
References

[1] Naranjo JM, Posada JA, Higuita JC, Cardona CA. Bioresource Technology Valorization of glycerol
through the production of biopolymers: The PHB case using Bacillus megaterium. Bioresour Technol
2013;133:3844. doi:10.1016/j.biortech.2013.01.129.
[2] Chen Y, Tsai P, Hsu C, Lee C. Enzyme and Microbial Technology Critical residues of class II PHA
synthase for expanding the substrate specificity and enhancing the biosynthesis of
polyhydroxyalkanoate. Enzyme Microb Technol 2014;56:606. doi:10.1016/j.enzmictec.2014.01.005.
[3] Li F, Zhang C, Liu Y, Liu D, Xia H, Chen S. Efficient production of ( R ) -3-hydroxybutyric acid by
Pseudomonas sp . DS1001a and its extracellular poly ( 3-hydroxybutyrate ) depolymerase. Process
Biochem 2016;51:36973. doi:10.1016/j.procbio.2015.12.016.
[4] Carlson R, Srienc F. Effects of recombinant precursor pathway variations on poly [( R ) -3-
hydroxybutyrate ] synthesis in Saccharomyces cerevisiae 2006;124:56173.
doi:10.1016/j.jbiotec.2006.01.035.
[5] Moncada J, Matallana LG, Cardona CA. Selection of Process Pathways for Biore fi nery Design Using
Optimization Tools: A Colombian Case for Conversion of Sugarcane Bagasse to Ethanol , Poly-3-
hydroxybutyrate ( PHB ), and Energy 2013. doi:10.1021/ie3019214.
[6] Dhanasekar R, Viruthagiri T SP. Sntesis de poli (3-hidroxi butirato) de una cepa mutante Azotobacter
vinelandii utilizando glucosa en un reactor discontinuo. 16. Diario de Ingeniera Bioqumica.; 2003.
[7] Marvi SP. Thermoplastic biopolymer production ( PHB , Poly 3-Hydroxybutyric acid ) using secondary
carbon source bioreactor based on nitrogen fixation biotechnology n.d.:2034.
[8] Lopez-arenas T, Gonzlez-contreras M, Anaya-reza O, Sales-cruz M. of the production process of PHB
( polyhydroxybutyrate ). Comput Chem Eng 2017. doi:10.1016/j.compchemeng.2017.03.009.
[9] Posada JA, Naranjo JM, Lpez JA, Higuita JC, Cardona CA. Design and analysis of poly-3-
hydroxybutyrate production processes from crude glycerol. Process Biochem 2011;46:3107.
doi:10.1016/j.procbio.2010.09.003.
[10] Mussatto SI, Moncada J, Roberto IC, Cardona CA. Bioresource Technology Techno-economic analysis
for brewer s spent grains use on a biorefinery concept: The Brazilian case. Bioresour Technol
2013;148:30210. doi:10.1016/j.biortech.2013.08.046.
[11] Akiyama M, Tsuge T, Doi Y. Environmental life cycle comparison of polyhydroxyalkanoates produced
from renewable carbon resources by bacterial fermentation 2003;80:18394. doi:10.1016/S0141-
3910(02)00400-7.