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What is polyhydroxyalkanoates (PHA)?

PHA is a family of biological polyesters that synthesized and stored as water insoluble inclusions in various microorganisms under nutrient-limited conditions in the presence of excessive carbon source.

What is differential scanning calorimetry (DSC)?

DSC is a thermoanalytical technique in which the difference in the amount of heat required to increase the temperature of a sample and reference are measured as a function of temperature. It can be used to observe fusion and crystallization events as well as glass transition temperatures (Tg). Besides Tg, melting temperature (Tm) and the enthalpy of fusion (∆Hm) of polyhydroxyalkanoates can also be determined.

What is the usage of mass spectrometry in the study of PHA?

Mass spectrometry is an analytical technique used to measure the mass-to-charge ratio of ions. It is most generally used to find the composition of a physical sample by generating a mass spectrum representing the masses of sample components. Thus, the isotopic composition of elements and the structure of PHA can be determined by observing its fragmentation.

What is the difference between one-stage cultivation and two-stage cultivation?

One-stage cultivation:  For growth-associated bacteria in PHA production.
Two-stage cultivation: For nongrowth-associated bacteria in PHA production.

Any difference between the PHA synthesized in eukaryotes and prokaryotes cells?

Yes. For instance, molecular weight of P(3HB) in eukaryotes is lower; 100-150 unit of 3HB monomer.

What is the role of P(3HB) in eukaryotes and prokaryotes cells?

Function of P(3HB) in Eukaryotes cells: involved in controlling the permeability of cell to minerals; Prokaryotes cells: As carbon and energy storage.

What it mean by inoculum in PHA production?

Inoculum is the seed culture for the PHA production; the amount of cells which is used to produce more cells (usually 3-10 % [v/v] in the culture medium).

: In PHA production, the first stage of fermentation is the growth phase; second stage means stop growth and the initiation of PHA accumulation; third stage is the PHA degradation phase.

: In fermentation, the first stage is the lag phase while the second stage is the log phase.

What it means by substrate specificity?

Enzyme that active towards certain substrates and has a low activity towards other substrates.

Can the wild type of Ralstonia eutropha produce mcl-PHA?

Substrate specificity of PHA synthase in R. eutropha is active towards C3, C4, C5 and 4HB; but a very small amount of C6, C8 and C10 monomers can also be incorporated.

How many types of PHA synthases can be distinguished?

With respect to the synthase substrate specificity and primary structures, there are 3 types of PHA synthase:

  • phaC (R. eutropha) that active towards short-chain-length (SCL) HA monomer which containing 3-5 carbon atoms.
  • phaC1 and phaC2 (Pseudomonas oleovorans) that efficiently incorporate medium-chain-length (MCL) (R)-3HA monomers which contain 6-14 carbon atoms.
  • phaC and phaE (cyanobacteria) which both subunits of synthase are necessary for the functional activity of the enzyme. The substrate specificity of PHA synthases in this group are apparently not very well defined, but generally they seem to prefer SCL HA.

What kind of dyes can be used to stain PHA inclusions in PHA-producing bacteria?

Sudan black B can be used to stain native PHA inclusions; Nile Blue A is more specifically staining the inclusions and exhibiting strong orange fluorescence; Nile Red can detects PHA directly in growing bacterial colonies.  SCL-PHAs absorp more Nile Red than MCL-PHA.

8 classes of biopolymer according to their chemical structure:

Nucleic acids: ribonucleic acids, deoxyribonucleic acids.
Polyamides: proteins and poly(amino acids)
Polysaccharides: cellulose, starch, xanthan.
Organic polyoxoesters: PHA, poly(malic acid), cutin
Polythioesters: recently reported.
Inorganic polyesters with polyphosphate.
Polyisoprenoids: natural rubber of Gutta Percha.
Polyphenols: lignin, humic acids.

How is the conversion of rpm to g-force?

g-force = 0.00001118 x radius of rotation (cm) x rpm2

What is the meaning of xenobiotic?

Xenobiotic means that something that is completely synthetic chemical compound and not naturally occurring on earth.

What is the suitable pH for the cultivation of cyanobacteria?

In open air, cultivation pH for cyanobacteria must be higher than 9.5 to inhibit the growth of other contaminant/bacteria. Anyway, the cost to maintain high pH is expensive.

How to maintain high pH in a cost effective way when culturing cyanobacteria?

Chalk (calcium carbonate) can be used; Spirullina will release bicarbonate into the medium and the medium will becomes alkaline automatically.

Less than 15 wt% of PHA content can be obtained by using cyanobacteria. Is that considers low?

Not really. The nutrient requirement is low for cyanobacteria; the PHA content may actually as high as bacteria, although wt% in cyanobacteria is low (cell size).

How is the quality of the polymer produced by cyanobacteria?

Comparatively is almost the same for the physical and thermal properties of PHA synthesized by bacteria.

Can cyanobacteria produce copolymer?

Probably yes, it can produce P(3HB-co-3HV), but usually it produce P(3HB) homopolymer.

What is polydispersity index?

Polydispersity index (PI) indicates the distribution of the polymer chain weight.
(PI > < 1 is considered good)

Why choose spirullina in PHA production?

It has been shown that spirullina able to produce PHA. Some of its advantages are:

  • during downstream process, the recovery of cell is easier compare to other bacteria; leave it to sendiment and no need to centrifuge.
  • Can be cultivated in a large scale.
  • Avoid using expensive carbon source in the PHA production (bacteria)
  • Spirullina is not toxic, compared to E. coli – pathogenic for human consumption as a health food.

(spirullina which accumulate PHA can also be consumed as a health food, it has been known it is not toxic or biocompatible).

Any effect of cultivation time to the molecular weight of the synthesized PHA?

PHA accumulation normally will take place for 24 hours, glass transition temperature is low. The longer the cultivation time (more than 72 h), Mw distribution, length of polymer will become shorter and shorter. If grow the cell for very long time, polydispersity index will become higher.

Essential criterion of PHA in tissue engineering:

A high biocompatibility is essential for the acceptance of an incorporated object by humans and mammals. Several factors to determine whether an object is biocompatible:

  • Shape
  • Surface porosity
  • Chemistry of the materials
  • Environment (tissue) where it is incorporated

(Traditionally used materials, e.g. silicone, are suspected to cause cancer. Thus, PHA has the potential to become an important compound for medical applications)

Five parameters that scaffold need to fulfill for successful tissue engineering:

  • Biocompatibility
  • Support of cell growth and cell adhesion
  • Guide and organize the cells
  • Allow ingrowth of cells and passage of nutrients and waste products
  • Biodegradable without formation of toxic compounds

What is thermoplastic?

A thermoplastic melts to a liquid when heated and freezes to a brittle state when cooled sufficiently. Thermoplastics are elastic and flexible above a glass transition temperature (Tm), can go through melting/freezing cycles repeatedly and they can be reshaped upon reheating.

Why mcl-PHA has a lower density compared to scl-PHA?

Side chain of MCL-PHA is much longer compared to SCL-PHA. Therefore it can’t be arranged in a compact manner inside the MCL-PHA granules. Thus MCL-PHA will has a lower density compared to SCL-PHA.