TRANSBIO. Valorización a través de fermentación sumergida.

BioTRANSformation of by-products from fruit and
vegetable processing industry into valuable
BIOproducts
This project has received funding from the European Union’s Seventh Framework Programme for research,
technological development and demonstration under grant agreement no 289603
ANA LÚCIA CAROLAS
HEAD OF LABORATORY – BIOTREND SA
Vitoria-Gasteiz, Spain, 03-10-2015
Biotrend SA Slide 0
TRANSBIO Workshop – Vitoria-Gasteiz, Spain– 3rd November 2015

Presentation outline
 Basics about submerged fermentation
 Development and process scale-up
 Example 1: production of PHB using naturally occurring strains
 Example 2: production of succinic acid using naturally occurring strains
Biotrend SA Slide 1

Chemistry of life
Biotrend SA Slide 2
Elements:
Carbon 50%
Oxygen 20%
Nitrogen 14%
Hydrogen 8%
Phosphorous 3%
Sulfur 1%
Potassium 1%
Sodium 1%
Other oligoelements
Main
cell compounds:
‐ Fats and lipids
‐ Polysaccharides
(cellulose, starch, etc)
‐ Nucleic acids
(DNA, RNA)
‐ Proteins
Sources:
CO2, sugars, proteins, fats
Air, water, medium components
Proteins, NH3 (amonia), NO3
‐ (nitrates)
Various culture medium components
PO4
3‐ (phosphates)
Proteins, SO4
2‐ (sulfates)
Various salts
Nutrients
Products
Cells
Cells

Kinetics of cell growth
Cell multiplication: Exponential cell growth…
timenumberofcells
1 2 4 8 … 2n This will only happen if no restrictions
to growth exist…
td td td
td: doubling time
Biotrend SA Slide 3

Cell multiplication
time
Lognumberofcells
lag phase
exponential
growth
stationary stage
death stage
Biotrend SA Slide 4

Bioreaction
A fermentation process is influenced by numerous factors, including:
- temperature,
- pH,
- composition of the medium,
- dissolved oxygen and carbon dioxide.
Most industrial fermentations are
carried out under highly controlled
conditions to maintain at
all times the most appropriate
conditions for the culture to grow
and/or to accumulate the desired
product. These optimum conditions
may be constant, or may vary over time.
O2
T
Cooling
water
pH
Acid
Base
pO2
Air
CO2
Exhaust
gasesNutrients O2
T
Cooling
water
pH
Acid
Base
pO2pO2
Air
CO2
Exhaust
gasesNutrients
Biotrend SA Slide 5

Initial development
 Carried out deep-well microplates or shake flasks at a scale lower than 1 L,
in incubators with control of temperature and agitation speed.
No control of pH or pO2
Many parameters/conditions tested at same time
 At this stage, the priority is to establish the basic parameters of the culture
conditions of the microorganism, such as the growth medium, tolerance to
inhibitors and first estimates of growth rates and yields.
Biotrend SA Slide 6

Small scale reactors
 Bioreactors at 1-10 L scales, equipped with monitoring instrumentation and
typically control of temperature, pH, dissolved oxygen, stirring speed, and
foam.
 At this scale, advanced fermentation strategies can be designed and tested
in order to increase the process productivity.
Biotrend SA Slide 7

Pilot scale reactors
 Normally with a capacity of up to 1000 L,
mimicking the industrial scale reactors, including:
 Same geometry
 Auxiliary equipment and utilities such as air
compressors, steam generators, chillers,
etc. and a set of monitoring sensors for the
reactor itself and for the appropriate
production and operation of the utilities.
Biotrend SA Slide 8

Modes of operation
 Batch: A batch of culture medium in the fermenter is inoculated with a
microbial culture. The fermentation proceeds for a certain duration and the
product is harvested.
 Fed-batch: Sterile culture medium is added either continuously or
periodically to the inoculated fermentation batch. The volume of the
fermenting broth increases with each addition of the medium. The
composition of the feeding medium may vary with time.
 Continuous: Sterile medium is fed continuously into a fermenter and the
fermented product is continuously withdrawn, so the fermentation volume
remains unchanged. Typically, continuous fermentations are started as
batch cultures and feeding begins after the microbial population has
reached a certain concentration.
Biotrend SA Slide 9

Stirred tank reactor
The stirred tank fermenter is the most
commonly used types because of its
flexibility.
It consists of a cylindrical vessel with a
working height-to-diameter ratio of 2–4
with a central shaft that supports
impellers for effective mixing.
Baffles (4)
Drive motor
Turbine
agitator
Air
Water/steam
jacket
Exhaust
gas
Air
sparger
Biotrend SA Slide 10

Challenges - Mixing
Efficient supply of oxygen is critical in aerobic fermentations:
 Ensure the unrestrained metabolic activity of the cell.
 Avoid diversion of carbon to unwanted products:
- Reducing yield
- Requiring their separation from the end product
- May rapidly accumulate to growth-inhibitory levels.
oxygenconcentration
10 L 10 m30
100

Challenges - Mixing
 During fed-batch cultures, the nutrient medium is
fed, typically from the top of the bioreactor.
 Some cells will be in contact with excess nutrient
while others will be in starving conditions.
 Excess nutrients may inhibit growth or divert
metabolism from one saturated reaction pathway
to another, eg. producing unwanted by-products,
thereby lowering the overall yield.
Feed
Nutrient conc.
Excess
substrate Growth and/or
desired product
By-products
Cell
Maximum flux through the pathway
that produces the desired product
0 100

Heat transfer efficiency
 When designing the bioreactor, the following heat loads must be
considered:
+ Heat released by the biological reaction (metabolism):
the production of heat by the culture is linearly correlated
to the consumption of oxygen
+ Heat dissipated by mixing
+ Air expansion from the bottom (higher pressure) to
the top of the bioreactor (lower pressure)
- Heat loss to the surrounding environment
 Normally, when performing a high-cell density fermentation, the fermenter
must be continuously cooled in order to compensate for the heat released
by the high metabolic activity and for the heat dissipated by mixing.

Inoculum generation
1 L 20 L 400 L 12000 L 240 m3
Seed
culture
Shake
flask
Seed fermenters
Production
fermenter
 A seed culture is used to inoculate sterile liquid medium in shake flasks.
 After sufficient growth, this pre-culture inoculates the “seed” fermenter.
 Since industrial fermenters can be quite large, the inoculum is built up
through successive stages, to 5–10% of the working volume of the
production fermenter, ensuring optimal use of the production fermenter by
minimizing the batch time in this vessel.

PHA’s
Polyhydroxyalkanoates (PHAs) are a class of bio-based polyesters with
attractive qualities for thermoprocessing applications.
PHAs are aliphatic polyesters produced via fermentation of renewable
feedstocks directly within the microorganism via fermentation.
The PHA accumulates as granules within the cells and serves as a microbial
energy reserve material.

PHA’s
A wide range of PHA homopolymers (repeats of the same monomer),
copolymers (repeats of two monomers), and terpolymers (repeats of three
monomers) have been produced, in most cases at laboratory scale.
The range of PHA structural architectures that is now accessible has opened
up a broad property space, encompassing rigid thermoplastics, thermoplastic
elastomers, as well as grades useful in waxes, adhesives, and binders.
P3HVP3HBPHA

PHA’s
Carbon sources
- sugars;
- glycerol (biodiesel
by-product);
- lignocellulosic
hydrolysates;
Biomass
build-upO2
CO2
CO2
PHA
accumulation
Nutrition medium with
limited N and/or P
O2
Normally, a biomass build-up stage is followed by nutrient limitation with
excess carbon source. This will trigger a stress response in the strain which
will then accumulate the carbon which is continuously fed in the form of PHA.

PHA’s
0.0#
0.5#
1.0#
1.5#
2.0#
2.5#
0# 10# 20# 30# 40# 50# 60# 70#
Produc'vity,(g/L.h),
2#L#
10#L#
50#L#
250#L#
Enhanced bioproduction of
poly-3-hydroxybutyrate from wheat straw
lignocellulosic hydrolysates
RESEARCH PAPER New Biotechnology Volume 31,Number 1 January 2014

Succinic acid
A platform chemical:
Solvents
Pharmaceuticals Pyrrolidones
Coatings and pigments
Polyurethane
Plasticizer
Freezing point
depression agents
Food
1,4-BDO/THF
PBS

Succinic acid
Bacteria: pH 6-7
+ Available succinic acid-producing strains
+ Genetic tools available (ex. E. coli)
- At the optimum pH, succinate was produced
instead of the free succinic acid. To obtain
the free acid, gypsum was produced as
by-product.
- Risk of infection by bacteriophages reflects
on higher capital expenditure.
Yeasts: pH ~3
- Succinic acid-producing strains to
be developed
+ Genetic tools available (ex. S.
cerevisiae)
+ At the optimum pH, the free
succinic acid is produced (no
gypsum production)
+ Robust organisms.
Many research groups and company cloned in yeast host genes of the
bacterial succinic acid pathway.
In TRANSBIO we are working with non genetically modified strains.

Succinic acid
 Screening in deep well plates:
- 870 non-Saccharomyces yeast strains
- 184 Saccharomyces spp
 Link metabolism to
fermentation strategy
- Supply of carboxylation
substrates
- Limiting biomass formation
- Limiting ethanol formation
Acetate
CO2
Phosphoenolpyruvate
Pyruvate Acetaldehyde
Ethanol
Acetyl-CoA
Oxaloacetate
Isocitrate
Malate
Fumarate
 -Ketoglutarate
Succinate Succinil-CoA
CoA
Citrate
CO2
Biomass
(Lipid, protein
DNA, RNA)
Acetyl-CoA
CO2
Biomass
(Lipid, protein,
DNA, RNA)
M itochondria
Cytosol
ADP
ATP
Pyruvate
NAD+
NADH
NAD+
NADH
NAD(P)+
NAD(P)H
ATP
AM
NAD+
NADH
CoA
NAD+
NADH
CO2 NAD+
NADH
CoA
ADPATPCoA
FAD
FADH2
Glyoxylate
CoA
CO2
ATP
ADP
NADH/FADH2
NAD+
/FAD
P/O*ADP
P/O*ATP
NAD+
NADH
C sources

Succinic acid
TB*1"
TB*2"
TB*3"
TB*4"
TB*5"
TB*6"
TB*7"
TB*8"
TB*9"
TB*10"
W372"
Ref.1"
Ref.2"
Ref.3"
W372, commercial strain as tested in TRANSBIO and used in Ref.1: J.-L. de Klerk (2010) Succinic
acid production by wine yeasts, Masters Thesis, Stellenbosch University, South Africa);
Ref. 2: Raab. Et al (2010) Metabolic Engineering 12, 518-525;
Ref. 3: Otero et al. (2013) PLoS ONE 8(1): e54144.

Succinic acid
• Tests on:
Pressed banana
pulp juice
Sweet corn
hydrolysate
Potato pulp
hydrolysates

Contacts:
Ana Lúcia Carolas
+351 231 410 940
ana_carolas@biotrend.biz
www.biotrend.biz

TRANSBIO. Valorización a través de fermentación sumergida.

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