Discussion:

Fermentation also referred as the bulk growth of microorganisms on a growth medium,

often with the goal of producing a specific chemical product. Polyhydroxyalkanoate
(PHA) is a polymer that is synthesized by bacteria as an intracellular carbon and energy
storage compound under nutrient-limiting conditions with excess carbon. The PHAs are
non-toxic, bicompatible, biodegradable thermoplastics that can be produced from
renewable sources. They have high degree of polymerization, highly crystalline and
insoluble in water. Among various known biodegradable polymeric materials, PHA is
known to be a fully biodegradable alternative to conventional plastics. This experiment
was done to synthesize PHA via batch fermentation using bioreactor. PHA biosynthesis
experiment was carried out using Cupriavidus sp. USMAHM13.
Among the poly(3-hydroxybutyrate-co-4-hydroxybutyrate), [P(3HB-co-4HB)]
accumulating bacteria, Cupriavidus sp. USMAHM13 was chosen as the subject of this
experiment due to its ability to convert 1,4-butanediol into high PHA content and 4HB
compositions. Biosynthesis of P(3HB-co-4HB) strongly depends on the use of precursor
substrates as carbon sources. 1,4-butanediol is among the most common and suitable
carbon precursors for biosynthesis of P(3HB-co-4HB) in Cupriavidus sp. USMAHM13.
This bacterium can able to convert acetyl-CoA to 3HB-CoA as it is able to metabolize
1,4-butanediol in order to synthesize copolymers containing 3HB and 4HB units.
Cupriavidus sp. USMAHM13 is an aerobic, motile and rod-shaped gram-negative
bacterium. This bacterium is believed to represent a novel strain in the genus Cupriavidus
because it exhibited yellow pigmentation. The crude yellow pigment consist of
antimicrobial property. The special properties of this bacterium are that it can produce
both scl-PHA and mcl-PHA. These characteristics of PHAs can be very useful in
applications like implants, gauzes, matrix material for slow release of drugs and so on.
Besides that, selective media has been used in this experiment to allow certain types of
organisms to grow and inhibit the growth of other organisms. There are many
components in mineral salt medium which promotes the growth of microbes. The
KH2PO4 and K2HPO4 in MSM act as a buffer. This is to control the acidity as carbon
dioxide will be released during microbial growth. Then nitrogen source ammonium
acetate NH4C2H3O2 has been added during the preparation of mineral salt medium. The
ammonium NH4+ influenced bacterial growth and PHA production as it was required by
microorganisms to synthesize all the enzymes that were directly involved in inducing
metabolic process in the cells.
Nutrient limitation is essential to trigger PHA accumulation and generally, ammonia was
employed as the critical control factor for uncoupling the growth of cells and PHA
production. Carbon source such as fructose and 1,4-butanediol has been converted into
biopolymer by the bacterium which they stored inside their cells as granules. The main
purpose of the carbon source for microorganisms is to provide energy and carbon
skeleton for the synthesis of various biological compounds. Then, the trace element has

been added into the flask. The trace element consist of Mn, Zn, Cu, Cl, Na and so on
which is for optimum growth of bacteria.
The bacterium was cultured into the nutrient rich broth medium and incubated on the
orbital shaker at 28C for 18-20 hours at the rate of 200 rpm. The growth of
microorganism in shake flask is a simple method of fermentation. The flask is shaken
during the cultivation to mix the cell and the media, increase the homogeneity between
these two and also to provide aeration for the cells. The bacterium was also found to
produce yellow pigment when cultivated in nutrient broth. Various parameters such as
temperature, pH and ratio of culture volume to flask volume were found to influence the
yellow pigment production. The results from the shake flask culture will be used for
bioreactor process.
Apart from that, batch fermentation was chosen as the fermentation mode for the PHA
biosynthesis. It is a closed system, where the reactor is filled with a sterile nutrient
substrate and inoculated with the microorganism. During the entire fermentation process,
nothing is added except oxygen (in the form of air), an antifoam agent, and acid or base
to control the pH. Four typical phases of growth are observed such as lag phase ,
exponential phase, stationary phase, and death phase. 5L benchtop mechanically agitated
stirred-tank bioreactor has used for the biosynthesis of PHA. The function of bioreactor is
to provide a controlled environment for the growth of microorganism or defined mixture
of microorganism to obtain a desired product. The stirred tank reactor is the most
commonly used type of reactor with internal mechanical agitation. The stirred tank
bioreactors provide high values of mass and heat transfer rates and excellent mixing.
A modern mechanically stirred tank bioreactors consist of an agitator system, an oxygen
delivering system, a foam control system, a temperature control system, a pH control
system, sampling ports, a cleaning and sterilization system and a sump and dump line for
emptying of the reactor. A bioreactor is divided in a working volume and a headspace
volume. The working volume is the fraction of the total volume taken up by the medium,
microbes, and gas bubbles. The remaining volume is called the headspace. For this
experiment, 2 L of working volume has been used.
The bench top models are made of glass with a stainless steel head-plate. The function of
agitation system is to provide good mixing and thus increase mass transfer rates through
the bulk liquid and bubble boundary layers as well as to provide the appropriate shear
conditions required for the breaking up of bubbles. The agitation system consist of baffles
and agitator. The baffles are used to break the liquid flow to increase turbulence and
mixing efficiency. Agitator will create a constant living conditions through homogeneous
distribution.
Furthermore, the air sparger helps to break the incoming air into small bubbles. Bubbles
rise directly beneath the impeller. Shear forces are highest around the impeller. This
maximizes efficiency of bubble break up. The impeller also stirs the culture and maintain
the homogenous of the culture.The headplate with O-ring actually prevents the air from
entering into the bioreactor and effective temperature control is achieved in fermentation

reactors by the use of heating coil,temperature probe and water jacket. The temperature
sensor will detect when there is an increase or decrease in the tempreature of the culture
and causes the water to flow through the water jacket to maintain the temperature.
Moreover, the compressed air is supplied from vacumm pump to the culture through
sparger. The amount of oxygen and the flow rate enters to the culture are controlled by
the rotameter. The oxygen enter the bioreactor from gas phase to liquid phase in culture
media and to solid phase in the cell.
Then, foam can be produced during microbial fermentation. Foaming may occur either
due to a medium component or due to some compound produced by the microorganism.
Excessive foam formation can lead to blocked air exit filters and cause pressure to build
up in the reactor which will lead to a loss of medium or damage to the reactor. Foam can
be controlled with aid of antifoaming agents based on silicone. The antifoam sensor is
fixed at the headplate to detect any foaming in between the process and the sensor is
connected to the antifoam solution. The antifoam solution will be released if there is any
foaming activity detected by the sensor to break the bubbles. Antifoams are surface active
agents where they reduce surface tension in the foams and destabilize the protein film.
The bioreactor provide control of pH via liquid reagent addition through a pump. The pH
probe has calibrated first before installation and sterilization. The pH value is
continuously monitored to keep the environment optimal for cell growth. Before undergo
the fermentation process, bioreactor and all its compartment as well as the glass vessel
containing MS medium has been sterilized first in autoclave to prevent contamination.
Autoclaving is the one of the technique used in moist heat sterilization to kill the
microorganism. Then, two -point calibration of dissolved oxygen is done after
autoclaving. Tight control of temperature, pH , oxygen content, feed consumption, liquid
evaporation and foam levels all contribute to a much higher biomass and better protein
yield. The bioreactor has run with the IRIS to capture the data. The system should be at
steady state before the addition of bacteria.
Once the probes are calibrated, agitation is stable at 200 rpm, temperature is at 37C and
pH is close to 6.8, distilled water, fructose, ammonium sulphate and trace element was
added into the glass vessel of bioreactor as nutrient to the bacterial cell. The amount of
carbon source added is higher compared to nitrogen source for the effective production of
PHA. Then,the inoculum has been added into the sterilized port through a syringe. The
bacterial cultures were incubated for 48 hours. Samples were taken for every 12 hours,
and the date and time were recorded. After 48 hours, the bioreactor was turned off and the
comprssed air supply was stopped. Downstream processing constitutes a key part of the
entire PHA production process. The culture was harvested. The MS medium containing
bacterial culture was transferred to a bottle and the bioreacter was washed and fixed back.
In order to analyze the growth and pigmentation profile of strain USMAHM13,
absorbance reading for the optical density is taken from the spectrophotometer. The
experiments were carried out in triplicate. The pigments were extracted by repeated
centrifugation until the freeze-dried cells turned colourless. Freeze-dried cells were added
to methanol as it functions as an antifreeze. The profile of the bacterial growth can be

studied by plotting cell dry weight(g/l) versus time. As for this experiment, the
absorbance and cell dry weight increases from 0 hours until 36 and then it starts to drop
from 36th hours to 48th hours. At 0 hours the cell dry weight was at zero. Then, cell dry
weight of 12.6g/L for 12th hour is obtained during lag phase. Lag phase is where the cells
are adjusting to their new environment. During this phase, cellular metabolism is
accelerated, resulting in rapid biosynthesis of cellular macromolecules and in preparation
for the next phase of the cycle. Then, cell dry weight of 38.1g/L for 24th hour is obtained
during exponential phase where the microorganisms are in rapidly growing and dividing
state. The growth medium is exploited at the maximal rate and the color the of the
medium turns to dark yellow which is due to the pigment produced by the Cupriavidus
sp. USMAHM13. Next, highest cell dry weight of 56.85g/L for 48th hour is obtained. At
this stage the cells are still in log phase as it keeps growing and multiplying. However at
48th hour the cell growth declined as the cell dry weight is 49.05g/L which is lower
compared to previous reading. This shows, the cells have reached its maximum growth
and stops to grow. The depletion of nutrients and the subsequent accumulation of
metabolic waste products and other toxic materials in the media will facilitates the
bacterium to move on the death phase. During this phase, the bacterium completely loses
its ability to reproduce.
The harvested culture was thawed and transfered into falcon tube which later centrifuged
to separate the culture into supernatant and pellet. The centrifugation of harvested culture
also helps to remove unmetabolized substrate. The supernatant was removed and the cells
were washed to remove any debris and pellet was transferred into a container. The pellet
was stored at -20C for further use.
Then on next day the pre-frozen samples was break into small pieces and transfer into the
freeze-drier bottles and install the bottles to the machine. It was lyophilized fully to
obtain the dry bacterial cell mass. The weight of crude polymer was 2.48gm. The freezedried cells were transferred into the schott duran bottle containing chloroform which
helps to disrupt the cell walls of the bacteria. Chloroform extraction is a very simple and
effective method to separate PHA granules from the biomass. By using this method,
highly purified PHA can be obtained without degradation of PHA molecules. Then, the
extract were filtered using filter paper to remove the cell debris. The filtrate was
transferred into rotary flask to separate the polymer from the chloroform. Then the
concentrated filtrate was purified using chilled methanol to precipitate the dissolved
polymer. Initially, phase separation into hydrophobic and hydrophilic layer is occurred
when use methanol towards the polymer. During this process clumps of polymers was
observed in the bottle. The precipitated polymer was recovered through filtration and let
it dried overnight. .On the following day, the crude polymer was dissolved in chloroform
and poured into the petri dish.Once the chloroform evaporated, formation of yellow film
could be observed.
A major benefit of PHAs is that they can be both produced and broken down by microorganisms in almost every environment. They are also totally degraded to water and
carbon dioxide under aerobic conditions and to methane under anerobic conditions by
micro-organisms in soil, lake water, sewages and sea water. Polyhydroxyalkanoates are

100% biodegradable polymers. In terms of its applications, PHA is a good material for
bioplastics and implant biomaterials due to its biodegradability, biocompatibility,
thermoprocessibility and sustainability. For example, medical purposes like preparation
of implants or surgical devices, PHAs are superior to conventional plastics due to their
biocompatibility.

Coclusion:
In summary, Cupriavidus sp. USMAHM13 a novel yellow-pigmented bacterium was
capable of producing the, P(3HB-co-4HB) with various monomer compositions through
batch fermentation process.The characteristics of the copolymers were not only
influenced by the 4HB composition, but also by the type of carbon and nitrogen sources
as well as their concentrations. The interaction of upstream and downstream processing is
very important for the production of Polyhydroxyalkanoates (PHAs). Sterilization is
necessary during fermentation process to prevent contamination with any undesired
microorganisms. The production of bioplastics such as PHAs are considered as
sustainable processes where use renewable resources and also produce no net additional
CO2 to the atmosphere.
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