DR. B.R.

AMBEDKAR NATIONAL INSTITUTE

OF TECHNOLOGY, JALANDHAR, PUNJAB

Bioprocess Modelling and Simulation

BTPC- 304
Project(Assignment-1)
Optimization of bioprocess parameters for the production of biofuels
using a process simulation approach

Submitted by:- Submitted to:-

Harsh Gupta (20111026) Dr. Prangya Ranjan Rout(Assistant Professor)

Kajal Jain (20111029)

Venika (20111057)

Shreya (20111055)
Contents:
1) Introduction
2) Importance of Biofuels as a sustainable alternative to fossil
fuels
3) The Media & strain (Some Information)
4) Defining the Ideal Ranges
5) The Bioreactor
6) The Mass Balance
7) General Industrial Flow
8) The Simulation Procedure
9) Bioprocess Parameters And their Simulation Results
1)Introduction:

This paper presents a brief analysis and optimization of the

various parameters that are used in the production of Biofuel using a
simulation approach.There are many biofuels that a could be studied and their
bioprocess parameters optimized,but here in this paper we have used Ethanol
as aur biofuel.

The Simulation software used throughout this paper is SuperPro

Designer(Trial Version) and only a single fermenter is used in it.In actual
practice many units are connected to get the overall product.The process also
includes downstream processing to get the optimized,Here we have refrained
from downstream processing.

The results were not as expected even after following the complete protocol
and processes. The reason accounted for this may the random values takes for
simulating the process as in the field, the values are taken according to the
experimental procedure and thereon the optimization is done.Another reason
that may be thought of is the partial usage knowledge of the software.
Although various resources have been used to study the software, the software
in itself is complex to handle.
2)Importance of Biofuels as a sustainable alternative to fossil fuels:

1. Renewable Energy Source: Biofuels are derived from organic matter, such as crops,
agricultural residues, algae, or waste biomass, which can be sustainably produced through
agricultural or industrial processes. Unlike fossil fuels, which are finite and depleting
resources, biofuels can be produced on a renewable basis, making them a potential long-
term solution to energy needs.
2. Reduced Greenhouse Gas Emissions: Biofuels have the potential to significantly reduce
greenhouse gas (GHG) emissions compared to fossil fuels. During their production and
combustion, biofuels generally release fewer net carbon dioxide (CO2) emissions because
the carbon released during combustion is offset by the carbon absorbed by the plants during
their growth. This can help mitigate climate change by reducing GHG emissions, which are
responsible for global warming.
3. Energy Security and Diversification: Biofuels can contribute to energy security by
reducing dependence on fossil fuel imports. Many countries rely heavily on imported fossil
fuels, which can be subject to price volatility, geopolitical tensions, and supply disruptions.
By developing domestic biofuel production, countries can diversify their energy sources
and reduce their vulnerability to external energy shocks.
4. Economic Benefits: Biofuel production can create economic opportunities by generating
jobs, stimulating rural economies, and supporting local agriculture. Biofuel production can
also provide a market for agricultural residues, waste biomass, or other organic materials
that would otherwise go unused, contributing to circular economy principles.
5. Technological Innovation: The development of biofuels as a sustainable alternative to
fossil fuels requires research and innovation in areas such as biomass production,
conversion technologies, and process optimization. This can drive technological
advancements, foster innovation, and spur the development of new industries and markets.
3)The Media & strain (Some Information):

The media used in a bioprocess for ethanol production from glucose using yeast as the
microorganism is typically formulated to provide the necessary nutrients for yeast growth
and metabolism. The composition of the media would depend on the specific requirements
of the yeast strain being used, as well as the process conditions and desired ethanol
production yield.

The media used in ethanol production processes generally contain a carbon source, such as
glucose, to serve as the substrate for yeast fermentation. Other components, such as
nitrogen sources (e.g., yeast extract, peptone, ammonium salts), minerals, vitamins, and
trace elements, may also be included to support yeast growth and metabolism. These
components can be included in the mass balance equations as additional components and
their consumption kinetics can be incorporated into the model.

The specific composition and formulation of the media varies acoording to the yeast strain, the
availability and cost of media components, and the desired process performance. It is
important to accurately represent the media composition to balance equations.
Experimental data on media composition and its impact on yeast growth and metabolism
can be used to determine the appropriate media mass balance equation and parameter
values for the bioprocess model. Here in our work we have taken water, carbon and glucose
as our media.

Microorganisms: Saccharomyces cerevisiae is a commonly used microorganism for ethanol

production. It is a yeast strain that is capable of fermenting various types of sugars,
including glucose, fructose, and sucrose, into ethanol. S. cerevisiae is a robust
microorganism that can tolerate a wide range of pH (typically pH 4-6) and temperature
(typically 25-35°C) conditions. Other yeast species, such as Candida spp., Pichia spp., and
Kluyveromyces spp., have also been used for ethanol production, depending on the specific
feedstock and process requirements.

No specific Strain of S.cerevasie was used ,instead the pre registerd Yeast was selected as
the microorganism.
4)Defining the Ideal Ranges:

1. Ethanol production: carbohydrate-rich feedstocks, such as sugars or starches, and you have
access to suitable strains of yeast or bacteria, ethanol production through fermentation
could be a viable option. Yeast, such as Saccharomyces cerevisiae, is a widely used
microorganism for ethanol production and can be relatively easy to cultivate. The growth
conditions for yeast are also relatively moderate in terms of temperature and pH
requirements.
2. Byproduct: Carbon dioxide (CO2) is typically produced as a byproduct in ethanol
production through fermentation.
3. Microorganism: Yeast (Saccharomyces cerevisiae) or bacteria (Zymomonas mobilis) are
commonly used microorganisms for ethanol production.
4. Growth conditions: Yeast or bacteria may require a temperature range of 25-35°C, pH
range of 4.0-6.0, and appropriate nutrient concentrations for optimal growth.
5. Yield: The yield of ethanol can vary depending on the strain of microorganism used, the
substrate concentration, and the fermentation conditions, but it typically ranges from 70-
90% of the theoretical maximum yield.
5)The Bioreactor:

1. Bioreactor Type: Stirred-tank bioreactor, (performed in fed Batch mode) also known as a
mechanically agitated bioreactor, is preferred for ethanol production using S. cerevisiae.
Stirred-tank bioreactors are commonly used in industrial settings due to their efficient
mixing capabilities and scalability. (We have chosen Fermentor in our simulation)
2. Bioreactor Size: The size of the bioreactor would depend on the production scale and
desired yield of ethanol. It can range from a few liters for laboratory-scale experiments to
several thousand liters for large-scale industrial production.(The size is pre specified in
SuperPro designer and was not changed)
3. Agitation Rate: The agitation rate, expressed in revolutions per minute (rpm), would
depend on the specific requirements of S. cerevisiae and the bioprocess conditions. It
would be optimized to ensure efficient mixing and distribution of oxygen, nutrients, and
microorganisms in the bioreactor, without causing any detrimental effects on the
microorganism's growth or viability.
4. Temperature: The optimal temperature for S. cerevisiae growth and ethanol production is
typically in the range of 30-35°C. Therefore, the bioreactor would be maintained at this
temperature using a heating/cooling system to provide an optimal environment for the
microorganism's metabolic activity.(37 C temperature was fed in to the system)
5. pH: The optimal pH for S. cerevisiae growth and ethanol production is typically in the
slightly acidic to neutral range of 4.0-6.0. Therefore, the pH of the bioreactor would be
monitored using pH sensors and adjusted using acid or base solutions to maintain the
desired pH level throughout the fermentation process.(pH was chosen as per the set value)
6)The Mass Balance:

Mass balance equation for glucose:

Rate of glucose input - Rate of glucose consumption = Rate of glucose accumulation

Mathematically, this can be expressed as:

Vin - Vout - μXV = d(Glucose)/dt

where:

Vin: Rate of glucose input (L/hr)

Vout: Rate of glucose output (L/hr), assuming there is no glucose output

μ: Specific growth rate of S. cerevisiae (hr^-1)

X: Concentration of S. cerevisiae cells (g/L)

V: Volume of the bioreactor (L)

d(Glucose)/dt: Rate of change of glucose concentration over time (g/L/hr)

Mass balance equation for ethanol:

Rate of ethanol production = Rate of ethanol accumulation

Mathematically, this can be expressed as:

Yield*μXV = d(Ethanol)/dt
where:

Yield: Ethanol yield coefficient, representing the amount of ethanol produced per unit of
glucose consumed (g/g)

μ: Specific growth rate of S. cerevisiae (hr^-1)

X: Concentration of S. cerevisiae cells (g/L)

V: Volume of the bioreactor (L)

d(Ethanol)/dt: Rate of change of ethanol concentration over time (g/L/hr)

Considering Glucose, Yeast, Media and Ethanol

Glucose mass balance equation:

d[G]/dt = -k1 * [G] * [Y] / (Km + [G]) - k2 * [G] * [X] / (Km + [G])

where d[G]/dt is the rate of change of glucose concentration over time, [G] is the glucose
concentration, [Y] is the yeast biomass concentration, [X] is the ethanol concentration, k1
and k2 are the rate constants for glucose consumption by yeast biomass and ethanol
production from glucose, respectively, Km is the Michaelis-Menten constant for glucose
uptake, and [X] is the yeast biomass concentration.

Ethanol mass balance equation:

d[X]/dt = k2 * [G] * [X] / (Km + [G]) - k3 * [X]

where d[X]/dt is the rate of change of yeast biomass concentration over time, [X] is the yeast
biomass concentration, k2 is the rate constant for ethanol production from glucose, and k3
is the rate constant for yeast biomass decay.
Yeast biomass mass balance equation:

d[Y]/dt = k1 * [G] * [Y] / (Km + [G]) - k4 * [Y]

where d[Y]/dt is the rate of change of yeast biomass concentration over time, [Y] is the yeast
biomass concentration, k1 is the rate constant for glucose consumption by yeast biomass,
and k4 is the rate constant for yeast biomass decay.

Media mass balance equation:

d[M]/dt = -k6 * [Y] / (Km + [Y])

where d[M]/dt is the rate of change of media concentration over time, [M] is the media
concentration, [Y] is the yeast biomass concentration, k6 is the rate constant for media
consumption by yeast biomass, and Km is the Michaelis-Menten constant for yeast biomass
media uptake.

Ethanol mass balance equation:

d[X]/dt = k2 * [G] * [X] / (Km + [G]) - k3 * [X]

where d[X]/dt is the rate of change of yeast biomass concentration over time, [X] is the yeast
biomass concentration, k2 is the rate constant for ethanol production from glucose, and k3
is the rate constant for yeast biomass decay.

These equations can be solved simultaneously using a numerical solver, such as Runge-Kutta
method or finite difference method, to obtain the time-dependent profiles of glucose,
ethanol, yeast biomass, nitrogen, and media concentrations during the ethanol production
process. The specific values of the rate constants, Michaelis-Menten constants, and initial
conditions would need to be determined through experimental data or literature
information, considering the specific yeast strain, media composition, and process
conditions used in the bioprocess.
7)General Industrial Flow

1. Feedstock: Ethanol can be produced from a variety of carbohydrate-rich feedstocks, such as

sugar cane, corn, wheat, sugarcane bagasse, and lignocellulosic biomass (e.g., crop
residues, wood chips). The choice of feedstock would depend on factors such as
availability, cost, and sustainability considerations. Lignocellulosic biomass is a promising
feedstock as it is abundant, low-cost, and does not compete with food production.
2. Pretreatment: Lignocellulosic biomass requires pretreatment to break down the complex
structure into fermentable sugars. Pretreatment methods include physical methods (e.g.,
milling, grinding), chemical methods (e.g., acid or alkali treatment), and biological
methods (e.g., enzymatic hydrolysis). Pretreatment is crucial to improve the accessibility of
cellulose and hemicellulose to enzymes during the subsequent enzymatic hydrolysis step.
3. Fermentation: Once the feedstock is pretreated and hydrolyzed into fermentable sugars, the
next step is fermentation. Yeast, typically S. cerevisiae, is inoculated into the fermentation
vessel along with the hydrolyzed sugars, and fermentation occurs in anaerobic conditions
The fermentation conditions, such as pH, temperature, agitation, and aeration, need to be
carefully controlled to optimize ethanol production. Generally, the optimal pH for S.
cerevisiae is around 4-6, and the temperature is around 30°C.
4. Distillation: After fermentation, the ethanol-containing broth is subjected to distillation to
separate ethanol from water and other impurities. Distillation is typically carried out using
a multi-stage distillation process, which requires heat energy for vaporization and
condensation of ethanol. This step results in the production of anhydrous ethanol, which
can be used as a biofuel.
5. Yield: The ethanol yield depends on various factors, such as the efficiency of hydrolysis,
fermentation efficiency, and the composition of the feedstock. Typically, the ethanol yield
from starch-based feedstocks, such as corn or wheat, can range from 90-95% of the
theoretical yield, while the yield from lignocellulosic feedstocks can vary widely
depending on the pretreatment and enzymatic hydrolysis efficiency.
6. Co-products: Ethanol production can also result in co-products, such as carbon dioxide
(CO2) gas and distiller's grains, which can be used as animal feed or as a source of other
value-added products. The utilization of co-products can enhance the sustainability and
economics of the ethanol production process.
8)The Simulation Procedure:

Step1:- Downloaded the Software-SuperPro Designer and Selected a suitable fermentor for a
batch process from the unit procedure option.

Step2: registered the Pure components for the production of ethanol

Step 3:- Placed a fermenter and made the input and output stream to the fermentor

Step 4: selected the operation to be performed on fermentor

Step 5:- selected the parameters for the input stream of air, media like amount, flow rate, setup
time.
Step 6:- Selected the operating condition, volume, reactions in the fermentor
Step 7:- Selected the Transfer-out conditions
Step 8:- Now selected the media stream and enter the Mass Composition(%) of the reactants

Step 9: Selectedoutput stream of ethanol and observed concentration of the ethanol produced.
8.Bioprocess Parameters And their Simulation Result s

Bioprocess parameters are critical factors that influence the production of biofuels through

bioprocessing, which involves the use of biological organisms, such as microorganisms or

enzymes, to convert renewable feedstocks into biofuels.

Here is the detailed study of various Bioprocess Parameters that affect the Biofuel Production.

1.Type of substrate used: Various types of substrates can be used for the production of

Biofuels but for ethanol production a variety of carbohydrate-rich feedstocks, such as

sugar cane, corn, wheat, sugarcane bagasse, and lignocellulosic biomass (e.g., crop

residues, wood chips) is used.

The choice of feedstock depends on factors such as availability, cost, and sustainability

considerations. Lignocellulosic biomass is a most used feedstock as it is abundant, low-

 cost, does not compete with food production .It is the second generation substrate for

biofuel production.

Generation of biofuels in basically divided into three categories:

1) First generation.First-generation biofuels: These are produced from food crops such as

corn, sugarcane, and vegetable oil. The primary advantage of first-generation biofuels is

that they can be produced using existing agricultural infrastructure. However, they have

several drawbacks, including the potential for competition with food production, land-use

changes that can lead to deforestation, and limited greenhouse gas emission reductions.

2) Second-generation biofuels: These are produced from non-food crops or waste materials

such as agricultural waste, forest residue, and algae. Second-generation biofuels are less

controversial than first-generation biofuels since they do not compete with food production,

but they still have some drawbacks, such as high production costs and the need for

significant technological improvements.

3) Third-generation biofuels: These are produced from algae and other microorganisms.

Third-generation biofuels have the potential to provide high yields, reduce greenhouse gas

emissions, and have a low impact on food production and land use. However, they are still

in the early stages of development and are not yet widely available.
 In our Simulation We have used Glucose as the carbon source as our substrate and yeast as the

growing microorganism.

2.Temperature: Temperature is a crucial parameter in bioprocessing, as it affects the growth,

activity, and metabolism of the biological organisms involved in biofuel production. Different

microorganisms or enzymes have specific temperature ranges at which they perform optimally.

For example, thermophilic microorganisms thrive at higher temperatures, while mesophilic

microorganisms prefer moderate temperatures.Ethanol production can be influenced by both

very high and very low hydrolysis temperatures. Ethanol yield tends to increase when hydrolysis

temperature is raised for a short period of time. Similarly, ethanol production also improves with

low hydrolysis temperature and increased hydrolysis time. This pattern could be due to the fact

that low temperature and short time may not convert cellulose into fermentable sugars, while

high temperature and longer time may convert fermentable sugars into non-fermentable

substances.The perfect temperature ranges between 20 C and 35 C.

To see the optimum temperature , the temperature was varied accordingly and

Simulation was done.

3. pH: pH, which refers to the acidity or alkalinity of a solution, also plays a significant role
in bioprocessing. The bioreactor’s or fermentation medium’s pH can affect the activity and
stability of microorganisms or enzymes involved in biofuel production.Typically, the ideal
pH range for fermentation mediums utilized in the production of bioethanol is between 4.5
and 5.5, and they contain varying concentrations of sugar.

4. Agitation Rate: The rate of agitation is a crucial factor in regulating the absorption of
nutrients from the fermentation broth into cells, as well as the removal of ethanol from cells
back into the broth. Higher rates of agitation tend to result in increased ethanol production,
enhanced sugar utilization, and decreased limitations on both ethanol and cells.In yeast cell
fermentation, the agitation rate is typically adjusted to fall between 150-200 rpm. Research
has indicated that by maintaining an agitation rate of 200 rpm over a cultivation period of
36 hours at 30°C, maximum ethanol production of approximately 23.10 g/L can be
achieved.

5..Fermentation Time: The growth of microorganisms during fermentation is highly

dependent on the duration of fermentation. Inadequate fermentation time can impede
proper growth of microorganisms, while excessively prolonged fermentation time can have
toxic effects on microbial growth, especially in batch fermentation. Batch fermentation
typically yields the highest ethanol concentration in the fermentation broth. However,
complete fermentation of ethanol can be achieved at a lower temperature by extending the
fermentation time, which ultimately results in a lower ethanol yield. Studies have
demonstrated that the fermentation process is typically preceded by rotation at either 120
rpm or 150 rpm for a duration of either 24 hours or 72 hours.

In Our Simulation The Fermentation time was varied from 24 to 72hours with little or no
significant results observed .

6.Inoculum Size: The Inoculum Size was varied from 6% to 12% of our Substrate
concentration and different results were obtained for different size.
 7..Substrate concentration: The concentration of the feedstock or substrate used in the
bioprocess can also significantly influence biofuel production. The availability and
concentration of the substrate can affect the growth, metabolism, and product formation by
the microorganisms or enzymes. Optimal substrate concentration needs to be maintained to
ensure efficient biofuel production.

Other factors:

8.Nutrient Concentration: Nutrient availability in the fermentation medium is critical for the
growth and metabolism of microorganisms or enzymes used in biofuel production.
Nutrients, such as carbon sources, nitrogen sources, phosphorus, and trace elements, are
essential for the growth and reproduction of microorganisms, and their availability can
significantly impact the biofuel production rate and yield. Optimizing nutrient
concentration in the fermentation medium is crucial for efficient biofuel production.

9.Oxygen availability: Oxygen is a critical factor in biofuel production for aerobic

microorganisms or enzymatic reactions that require oxygen. Oxygen availability can affect
the growth, metabolism, and yield of biofuels. Oxygen supply methods, such as aeration or
oxygenation, need to be optimized to meet the oxygen requirements of the microorganisms
or enzymes involved in biofuel production.

Fig:- different amount of ethanol produced with different strain of yeast

Similarly various inputs of substrate concentration have been given and observed the
concentration of ethanol produced.

Fig:- Yeast concentration= 15.34 g/L and Glucose concentration= 150.41 g/L

Fig:- ethanol concentration generated= 2.54 g/L

Now simulated the production of ethanol by keeping the substrate concentration constant
and varying the concentration of yeast .

Fig:- yeast concentration= 7.53 g/L and substrate concentration 150 g/L
 - Fig:-
Ethanol produced in the output stream= 2.534 g/L

Fig:- yeast concentration= 22.5 g/L and substrate concentration 150 g/L
 Fig:- Ethanol produced in the output stream= 2.54 g/L

References:

Biofuels: Production, Applications, and Environmental Impact by David M. Mousdale,

Bioprocessing for Value-Added Products from Renewable Resources" by Shang-Tian Yang,

and "Principles of Fermentation Technology" by Peter F. Stanbury.

BIOETHANOL PRODUCTION FROM Chlamydomonas reinhardtii AND ITS

BIOPROCESS DESIGN VIA SUPERPRO DESIGNER by Sanem BULUT, İlayda

HIRAOĞLU and Ozancan KOÇAK