Advanced Fermentation in

Bioprocess Technology
Dwini Normayulisa Putri
Department of Chemical Engineering, Universitas Indonesia

4 April 2023
Universitas Singaperbangsa Karawang
Basic Concept of
Fermentation
Basic Concept of Fermentation
 Fermentation is a process which involves the
use of microorganisms for production of
compounds that have applications in the energy,
material, pharmaceutical, chemical and food
industries.
 In simple words, fermentation is a process used
to produce a specific product by living organisms.
 Examples of simple fermentation products such
as baker’s yeast and alcohols, as well as
complex fermentation products such as
therapeutic proteins, antibiotics, enzymes,
and genetically engineered materials
https://www.embibe.com/exams/fermentation/
Types of Fermentation

Fermentation Solid Subme

process occurs Fermentation
State rged process occurs
on solid
substrate in
Ferme Ferme in liquid
the absence or ntation ntation nutrient broth
minimum (SSF) (SmF) which rich with
water content water
Solid State Fermentation (SSF)
 SSF processes are those
microbiological processes in which
growth and product formation
takes place on and inside the
humidified solid substrate
 SSFs are usually used for the
fermentation of agricultural products
or foods. Some food fermentations
involving SSF: Soybean by Rhizopus
(tempe), Soybean meal by
Neurospora (oncom), Cassava by https://www.youtube.com/bioresource
Saccharomyces (tapai)
Factors Affecting SSF

Type of Type of
Inoculum size
microorganism substrate

Moisture and
Temperature
water activity
Application of SSF
Enzyme production
• protease, lipase, cellulose, pectinase, phytase, L-glutaminase, amylase, ligninase,
xylanase, etc
Organic acid production
• lactic acid and gallic acid

Secondary metabolite production

• gibberellic acids, aroma production, antibiotics

Poly-gamma glutamate production

Poly unsaturated fatty acid production

Advantages and Limitations of SSF
Advantages Limitations
 Higher product titer  Difficult to control pH
 Low capital and operating expenditure
 Difficult to control moisture
 Low waste water production content
 Low energy requirements
 Limited types of microorganism
 Simple and highly reproducible
can be used
 Simpler fermentation media
 Less space requirement
 Easier aeration
 Economic to use
 Lower cost of downstream processing
Submerged Fermentation (SmF)
 Submerged fermentation are those microbiological processes in which
growth and product formation take place using substrate present in
liquid form
 Industrial enzymes/other products can be produced using this process

(Source: Chiang et al., 2020)

Modes of SmF
 Batch  Fed-batch  Continuous
 Nutrients medium  Nutrients medium  The organisms are
are added into the are added into the fed with fresh
vessel at the start of vessel at the start nutrients while
the fermentation and in between removing the spent
process and nothing during the medium and cells at
is added in between fermentation process the same rate
the process
Advantages of SmF
 Batch  Fed-batch  Continuous
 Simple to use  Organism’s growth  Feed flow rate could
 Operability and rate and subsequent be optimized to
reliability oxygen demand could improve the
be controlled productivity and
 Lower chances of
 High cell density of growth rate
contamination
the cells could be  Longer period of
 Can be handled by
achieved productivity
relatively
 Increased production  High cell density of
inexperienced
operator of non-growth related cells could be
metabolites could be achieved
achieved
Factors Affecting SmF

Microorganism Medium
Inoculum size
strain components

Agitation and
pH Temperature
aeration
SSF vs SmF

(Source: Saran et al., 2019)

Advanced
Fermentation
Technology
During the last few years, fermentation technologies have
advanced in order to improve the quality and quantity of the final
product to meet industrial needs
 Concepts of the seven factors affecting
the competitiveness of bacterial
fermentation are presented in each circle.

Factor 1 represents the performance

metrics (i.e., titer, yield, and productivity)
that directly affects the competitiveness
of bacterial fermentation.

Factors 2–5 represent carbon sources,

fermentation medium, fermentation
mode, and substrate feeding,
respectively, which determine the overall
cell growth and target product production
performance.

Factors 6 and 7 represent the

fermentation scale-up and downstream
processes, respectively, which are the key
factors to consider for successful
industrialization of bioprocesses. Detailed
explanation for each factor is discussed
in the corresponding section in the main
text. Abbreviations: CM, controller model;
GRAS, generally recognized as safe; PM,
process model.

(Source: Lee et al., 2022)

Factor 1: Performance Metrics
 For competitive microbial fermentation, key
performance metrics (titer, yield, and
productivity) should first be defined, depending
on the target product
 The defined performance metrics should be
satisfied not only at a laboratory (lab)-scale, but
also at subsequent pilot- and demonstration
(demo)plant-scale
Factor 2: Carbon Sources
 Carbon substrates used in fermentation should be
determined at an early stage in a bioprocess because
they affect both microbial production performance and
process cost
 This problem becomes especially important in large-
scale fermentation, which is difficult to operate using
purified carbon sources (e.g., glucose) due to the high
cost.
 Carbon sources should be carefully selected among the
most abundant, least expensive candidates including
crude carbon sources (e.g., carbohydrates,
lignocelluloses, protein hydrolysates, fatty acids derived
from biomass crops, forest/animal waste, and food
waste).
Factor 3: Fermentation Medium
 Upon selection of carbon sources, a
fermentation medium should be carefully
designed because it is not only directly
linked with the fermentation
performance, but also with the costs of a
medium itself and downstream processes
(i.e., separation and purification)
 However, optimizing fermentation
medium is challenging because of a
production host’s metabolic complexity
(e.g., auxotrophy) and a number of
process variables to consider
Factor 4: Fermentation Mode
 Fermentation mode should be selected based on a target product and
production host, together with objective performance metrics
 Fermentation mode can be largely classified into batch, fed-batch, and
continuous fermentations

Factor 5: Substrate Feeding
 During fed-batch fermentation, which is the most frequently used
fermentation mode in industry, precise and real-time monitoring of
cultivation parameters, such as DO level, pH, CO2 evolution rate, and the
concentrations of cells, nutritional components, products, and byproducts is
important. Based on the results of test fermentations monitoring all these
parameters, an optimal feeding strategy can be designed.

Factor 6: Fermentation Scale-Up
 Once the lab-scale fermentation process is established, fermentation scale-up
studies are performed using larger bioreactors, as there are a number of
factors affecting fermentation performances during the scale-up.
Factor 7: Downstream Process
 In bio-based production of chemicals, downstream processes account for 20–
50% of total production costs
 In addition to developing more efficient and less costly downstream
processes, much effort has been exerted to minimize downstream processing
costs through strain development by systems metabolic engineering

Development of a bioprocess for succinic acid overproduction
Succinic acid is a four-carbon dicarboxylic acid widely used as a monomer for various
polymers and as a precursor for many different industrial commodity chemicals. Several
previous studies are presented here, which showcase the implementation of the seven
factors discussed in this review that affect the competitiveness of bacterial fermentation
(Figure I).

First, E. coli was initially selected as a host strain because it is a model organism with
various available genome engineering and cultivation techniques and has been widely
used for the production of various chemicals. Using an engineered E. coli, dual-phase
fermentation was conducted for succinic acid production where high cell density was
achieved during the first aerobic phase, followed by enhanced production of succinic acid
during the subsequent anaerobic phase (step 1 in Figure I)
[80,112]. To further increase succinic acid production, the biosynthetic pathways that
generate byproducts were deleted and metabolic flux distribution was optimized by
overexpressing the native mdh and Rhizobium etli pyc genes (step 2 in Figure I) [112].
However, despite the notable achievements, E. coli strains did not meet the criteria
necessary for industrial-level production.

In the second process, M. succiniciproducens, a facultative anaerobic bacterium that

naturally produces succinic acid, was used as a production host (step 3 in Figure I). To
increase succinic acid production, metabolic pathways generating byproducts were
deactivated by deleting the ldhA, pflB, pta, and ackA genes, resulting in the LPK7 strain
[113]. Metabolic flux toward succinic acid was subsequently increased by restoring pflB
gene (PALK strain) and overexpressing C. glutamicum mdh gene (PALKcgmdh strain; step
4 in Figure I) [10].

Using the constructed strain as a basis, various attempts were made for optimal
fermentation. For example, the Methylobacterium extorquens fdh gene was overexpressed
in the LPK7 strain to efficiently use formate (which can be derived from carbon dioxide) in
addition to glucose as carbon sources (step 5 in Figure I) [49]. In another study, the fruA
gene was removed or the E. coli glpK gene was overexpressed in the PALK strain in order
to efficiently utilize sucrose and glycerol as dual carbon sources [76]. During fed-batch
fermentation, residual concentrations of sucrose and glycerol were monitored to apply
exponential feeding, so that the concentrations of sucrose and glycerol were kept below 20
and 5 g/l, respectively (step 7 in Figure I) [76]. As to the fermentation medium, a
chemically defined medium tailored for M. succiniciproducens was developed by analyzing
the genomic features (step 6 in Figure I) [114]. To achieve higher succinic acid
productivity, high inoculum fed-batch fermentation [10] and membrane cell recycling
bioreactor (MCRB) system [76] were performed (step 8 in Figure I). Finally, the engineered
M. succiniciproducens strain could produce nearly homo-succinic acid without
byproducts, which simplified the downstream recovery and purification processes (step 9
in Figure I) [115,116].
References
 Saran, S., Malaviya, A., & Chaubey, A. (2019). Introduction, scope and
significance of fermentation technology. high value fermentation products:
human health, 1, 1-25.
 Lee, J. A., Kim, H. U., Na, J. G., Ko, Y. S., Cho, J. S., & Lee, S. Y. (2022).
Factors affecting the competitiveness of bacterial fermentation. Trends in
Biotechnology.