Team:Nantes/bioplastics
Bioplastics
Définition
A bioplastic is a plastic that is derived - partially or totally - from renewable resources and that can be degraded. They are high molecules composed of proteins, polysaccharides, lipids, or polyester depending on the source.
There are different types of bioplastics:
- Compostable plastic: the material can be degraded under certain conditions or by
particular
organisms.
- Photodegradable plastic: the polymers of the plastic are degraded by photo-oxidation.
Ultraviolet rays attack the groups that separate to form free radicals. These will then
interact with oxygen.
- Bio-sourced plastics: These plastics are produced from organic materials. The polymers can
be produced by microorganisms (MO), macroalgae, or plants.
Not all bio-sourced plastics
are
biodegradable in the environment: decomposition times are indeed variable.
Biodegradable plastics from the oil industry: these plastics are composed of polymers from oil but can be degraded by the environment.
Polyhydroxyalkanoate or PHA are naturally synthesized by some microorganisms.
They can be
synthesized from petrol oil, lactic acid, or other carbohydrates derived from compost or
algae.
Context :
To find an alternative to non-biodegradable plastics derived from fossil resources, we wish
to produce biodegradable and bio-sourced bioplastic from the polyhydroxyalkanoate (PHA)
family.
The algae Ulva spp washed up on the beaches and responsible for the green tides
would be the source of carbon for the production of these PHA bioplastics.
The process of creating biodegradable and bio-sourced bioplastics, produced by marine
bacteria, has already been implemented a few years ago in Brittany, using fruit and
vegetable detritus as raw material (Bruzaud, 2015).
Our ambition would be to achieve a
similar final product, using decomposing Ulva substrates and specific microorganisms.
The
carbonaceous material of Ulva contains complex sugars (mainly xylose and rhamnose).
These
sugars can be used by specific marine bacteria or archaea to synthesize
polyhydroxyalkanoates or PHAs.
We can also create recombinant bacteria using synthetic
biology tools to produce PHA bioplastics.
Several bacterial strains are capable of producing different types of PHAs. Such a variety
is intriguing, as it gives the choice of the bacteria-carbon substrate pair to be optimized,
depending on the specificities of each, to obtain the best PHA yield (Bruzaud, 2015).
These
PHA pellets can ultimately be used industrially to produce plastic components in different
applications mainly medical, pharmaceutical and biotechnological.
Objectives :
- To explore the possibility to produce a plastic that is bio-sourced and biodegradable
- To compare the various types of PHA bioplastics
- To valorize the Ulva algae responsible for green tides as a source of carbon for the
production of bioplastic
- To understand the different types of microorganisms which can produce bioplastics
- To make genetically engineered microorganisms for the production of bioplastics
Scientific explanations
Polyhydroxyalkanoate
Structure of polyhydroxyalkanoates or PHAs :
PHAs are hydroxyalkanoate polyesters composed of an aliphatic chain of carbon, oxygen, and
hydrogen.
The side chain is made up of several various repeated monomers, thus
determining the identity of the polyester.
There is a variety of PHAs.
There are 3 main categories of PHAs according to the size of the monomers:
- Short-chain length monomers (SCLs): from 3 to 5 carbon atoms.
- Medium-chain Length monomers (MCLs): from 6 to 15 carbon atoms and many functions
(halogen,
hydroxy...)
- Long-chain length monomers (LCLs): more than 15 carbon atoms.
While SCLs have rather rigid and brittle properties, MCLs and LCLs have rather elastic
properties.
Polyhydroxybutyrate or PHB belongs to the PHA class.
This polyester is less easy to
transform into a product because it is stiff and brittle.
Indeed, it has a high degree
of
crystallinity and a melting temperature of 175°C, too close to the decomposition
temperature.
The most common form of PHB is poly-3-hydroxybutyrate or P3HB.
Polyhydroxybutyrate-co-hydroxyvalerate or PHBV is a co-polymer of PHB.
The addition of
the
3-hydroxyvalerate (3HV) unit disrupts the highly crystalline structure of PHB and gives the
polymer improved mechanical properties, as well as a faster degradation rate.
The
mechanical
properties of this plastic are interesting because it has better elasticity compared to the
PHB bioplastic.
These elastic properties are still limited because if they allow the
plastic
to be less brittle, the plastic remains a little malleable.
Thus, the applications are
particular: we often use PHBV to manufacture medicines boxes or bottles, clothing, and other
medical accessories; but also in the industrial field to replace the conventional plastic
food packaging.
Synthesis of PHAs:
PHAs can be synthesized by bacteria or alternatively archaea and are stored in the cytoplasm
of these microorganisms as granules (Albuquerque et al; 2018).
These granules are
surrounded
by a phospholipid membrane and proteins, PHasins.
These PHasins proteins influence the
structure and number of PHAs within the granule.
The production of a particular variety
of
PHAs depends on the source of carbon, provided in the medium, as well as the microorganisms
and their metabolism pathways.
After comparing the different PHAs and their properties, we chose to focus on the synthesis
of PHBV.
Carbonaceous matter of Ulva as a substrate
Macroalgae are a renewable source of carbon for PHA production that does not require
field cultivation.
Thus, it is an easily exploitable source, especially during the warm
seasons, when algae biomass is preponderant.
In recent years, red and brown algae have already been used as a carbon source to
produce PHAs (El-Malek et al, 2020).
Green macroalgae have been little exploited for the production of PHAs, due to their
original composition in sugars. The polysaccharides composing the wall of sea lettuce
are indeed uncommon.
However, Ulva spp. are among the most attractive biomasses, as they
are abundant all over the world and have a high polysaccharide content.
The use of green
algae stranded during green tides as a substrate allows to combine recycling and
valorization.
The carbonaceous material of Ulvan (Ulva wall) is mainly composed of
xylose and rhamnose polysaccharides.
We wish to extract these sugars from the Ulva wall to use them as nutrients for the
growth of microorganisms.
Microorganisms capable of synthesizing PHAs
A few marine bacterial strains are capable of synthesizing PHAs from Ulva spp. The bacterial
strains Alteromonas, and Cobetia are of particular interest, as they can produce PHA
polymers due to the harsh marine conditions in which they live, i.e., high salinity, low
temperature, and low organic matter (Gnaim and al; 2021).
The first possible approach is
the
combination of two marine bacteria: Altermonas and Cobetia to optimize the synthesis of PHAs
from Ulva spp.
Indeed, a marine archaea Haloferax mediterranei has demonstrated its ability to produce both
PHB and PHBV from green algae, using Ulva sugars: rhamnose and xylose, but also from other
sugars.
This microorganism can live at high salinity levels.
Thus, halophilic microorganisms could be particularly interesting for the synthesis of PHAs.
However, all these bacterial species, as well as archaea, require specific culture
conditions, less obvious to set up in the laboratory.
That’s why to optimize our
protocol we
propose to modify an Escherichia coli bacteria by adding key enzymes of the PHA synthetic
pathway. E. Coli is more easily usable in laboratory conditions.
Our bacteria will be
modified by transformation with plasmids containing a biobrick and gene coding for PHA
synthase and PHAsin.
These enzymes were chosen by their ability to reuse our sugars
derived
from Ulva.
The figure presented after is an extract of the wiki of the Edinburgh team (Team: Edinburgh
OG/Model - 2018.igem.org) and demonstrates the more important enzymes of the pathway:
B-ketothiolase, Acetoacetyl-CoA reductase, and PHB synthesis.
Acetyl-CoA is easy to
produce
and the steps to converse PHA in PHBV are relatively easy too.
Hence, it makes sense to
look
for ways to improve the transition from acetyl-CoA to PHA.
Consequently, to upregulate
the
pathway we would increase their expression in cells.
Experimentations
As we said before, we propose a protocol to perform a modified BL21 E. Coli capable of
synthesizing PHBV from our sugar.
In the next paragraphs, we develop a genetic
engineering
process based on the utilization of biobricks from the iGEM database and a discontinuous
culture procedure of our bacteria to produce PHBV.
Finally, we also describe cell lysis
and
purification protocol.
Genetic engineering protocol of the transformation of BL21 E. Coli in a bacteria able to synthesize PHBV.
We start from the Biobrick BBa_K2260002 designed by iGEM Team Calgary 2017 and perform some
modifications of the promoter and the TAG.
We want to improve this system so as to
express
and upregulate crucial enzymes of the PHA metabolism pathway in BL21 E. Coli.
To improve
Calgary's Biobrick we focused on two levels: the promoter and the Tag.
Firstly, based on the ‘Principes de Génie Génétique’ written by Primrose, Twyman, and Old,
the T7 promoter needs to be closer to the start codon, as a rule, 9 nucleotides or less.
Presumably, this too-long length between the T7 promoter and the start codon had caused
inconclusive results.
That’s why we suggest placing the part BBa_0034 behind the T7
promoter.
In addition, we didn't choose the classical 6x Histidine but the 10x Histidine presented by
Part: BBa K844000 – parts.igem.org.
As is described in the Utah presentation, the 10X
Histidine gives the best affinity to the resin column.
Lastly, to be more consistent, we select the same plasmid for the biobrick than for the
other manipulations, that is pET11a.
This standardization permits their use in the
production of PHBV without Biobrick as a negative control.
Also, to determine the
weightings
of each PHBV production enzyme, we can test each enzyme one by one. Except that, we use the
same Bacteria as Calgary named E. coli BL21.
→ Our goal is to upregulate the PHBV pathway in a recombinant BL21 E. coli in order to
maximize the yield production of PHBV.
Step 1: Modification of the plasmid pET11a
Team Calgary 2017 proposed in their biobrick to put the part BBa_B0034 downstream of the T7
promoter. However, the sequence contains 12 nucleic acids and can disturb the transcription
of the sequence (Primrose, Twyman, Old, 2012). Thats’ why we propose to perform a Quick
Change protocol to add the biobrick sequences upstream of the T7 promoter. Primers are
designed with a floating tail containing the sequence of the biobrick and go top to tail in
order to permit the replication of the plasmids during PCR cycles.
We design the insert with beta-ketoacyl-ACP reductase (EC.1.1.1.36) but this is the same for two others, found in Cobetia marina.
Afterward, BL21 E. Coli would be treated with TSS and transformed with our modified pET11
plasmids. Bacteria would be put in culture overnight at 37°C with ampicillin. The next day a
liquid culture would be prepared at 37°C with ampicillin.
Minipreparation of plasmids would be performed to isolate our modified pET11a and we would
have to verify the integrity of the sequence by sequencing.
Bacteria that have the good
plasmid (with the biobrick and without other mutation) would be put in another medium and
some bacteria would be stored at -80°C with glycerol.
Step 2: PCR cloning to isolate our genes of interest from the DNA matrix.
In this second step, we isolate our genes by PCR on the whole genome of Haloferax
mediterranei or another organism that expresses these enzymes.
Primers used for this
step
begin at the ATG to the 20th nucleic acid of the coding sequence of the gene and a 5’
floating tail containing Nde1 restriction sites.
On the 3’ side, as we did before, the
primer starts the 20th nucleic acid before the stop codon and a floating tail containing the
BamH1 restriction site and a His10Tag.
After the PCR, electrophoresis would be done to
verify the good amplification of our insert.
The band isolated from the slice would be
purified and the concentration of DNA would be determined by Nanodrop.
Step 3: Digestion of our plasmids and insert
Modified pET11a would be recovered from our BL21 bacteria by minipreparation and digested
with Nde1 and BamH1. The same enzymes would be used for our insert.
Fragments of
interest
would be isolated by electrophoresis and purified before the ligation.
Competent BL21 bacteria would be made with TSS and we would inoculate recombinant plasmids.
After one hour, we would spread bacteria in ampicillin supplemented agarose slice.
Bacteria would grow overnight at 37 °C with ampicillin.
The day after, bacteria are put in liquid culture overnight at 37°C with ampicillin.
Step 5: Verifications
Plasmids would be purified from bacteria by Minipreparation and analyzed by PCR the success
of the recombination.
We would sequence the insert so as to verify the integrity of the
sequence.
Representation of our recombinant modified pET11a plasmid.
We precise that, so as to express all the enzymes required for the PHA pathway, we would need to perform three protocols of transformation one after another until we would have three plasmids coding for each enzyme in the same bacteria.
Step 6: Good transformed bacteria would be separated into three groups :
The first group:
Adding glycerol to this group for it to be stored at -80°C freezer,
The second group would be cultured in an LB medium with ampicillin overnight which will
allow
the production of enzymes.
The third group would be cultured in an enriched medium with ampicillin to start the
production
of PHBV (see the discontinuous culture procedure)
Step 7: Collect enzymes
We propose here to determine the yield production of our enzymes. With the second plate of
bacteria, we would lyse cells and purify enzymes on a Nickel or a Cobalt purification
column.
Charges will catch the flag and can easily isolate our proteins of interest.
We would
recover
our proteins by elution.
To dose the amount of proteins we suggest a semi-quantitative
Western
Blot with actin as a reference to compare the expression of our enzymes.
A
polyacrylamide slice
would be done and after the migration of the samples, we would transfer the bands on a
nitrocellulose membrane.
A first incubation of the membrane with an antibody anti-actin
would
determine the base expression and the next incubation with an antibody anti-His would let us
determine the expression of our enzymes.
Discontinuous culture procedure
We propose in the next step a batch culture before purification. The production of PHBV requires a high-density culture with 2 or 3 cycles of fermentation.
Firstly, bacteria are cultured in an enriched medium and grow exponentially.
When
exponential
growth is achieved, bacteria are moved in another bioreactor with a strict medium.
This
medium
is less nutritive than the first one and contains mainly sugar from our algae: xylose and
rhamnose particularly.
In consequence, cells will be stimulated and start to form stocks
with
sugar.
These stocks are PHBV.
However, we had upregulated the pathway so the
exponential
accumulation in cells would break the cell membrane and freed PHA granules in the medium.
The
two next paragraphs detail the composition of mediums based on protocol established by
Tatiana
Thomas (2019).
The first culture medium called “enriched” is composed of carbon from our algae at 10g/L
plus 1
g/L of Tryptone, yeast extract at 0.5 g/L, and sea salt.
The pH of this medium must be
neutral
so around 7,5.
The pre-culture of bacteria will grow at 37°C under agitation at 200rpm
and cell
growth is measured by spectrophotometry at OD=600nm to determine the exponential part.
Bacteria
in the exponential part will be transferred into the strict medium.
The second one led to the production and the release of PHBV in the culture medium.
The
composition is similar but not required Tryptone and yeast extract. Moreover, we increase
the
proportion of our sugars.
To summarize, we can find in the strict medium carbon sources
from our
algae at 20g/L, sea salt, and pH 7-8. We can, at this step, restart a cycle of fermentation
to
improve the yield of PHBV or add non-stop carbon sources.
The culture in this medium is
controlled by spectrophotometry at OD=600 nm every 30 minutes to control the death of
bacteria
by the accumulation of PHA.
We can use a more precise technique with Nil Red and a UV
Lamp.
The
Nil red will be attached to the PHA granules and the fluorescent signal at 635 nm will
increase
during the culture in the strict medium.
To prevent the degradation of PHA granules by
cells if
the reaction is too important we need to add an inhibitor of PHA degradase (pasteurization
at
80°C for 15 min is effective).
Purification
Eventually, we propose to purify PHA from our culture to add chloroform and heat the mix at
60°C
to break all bacteria membranes remaining.
Next, the solution is centrifuged at 5000 rpm
for 7
min and the supernatant is passed through a cellulose acetate filter of 0.2 µm.
The
solution
obtained is finally left on the bench until the chloroform is fully evaporated.
Limites et risques
Application of our protocol
All protocols we presented below are the results of bibliographic work and the analysis of
different strategies of PHA production. Unfortunately, we didn’t have the possibility to
test our protocols and consequently to correct and adapt according to experimental results
our protocols.
Furthermore, we did not perform accurate modeling to determine the weight and
regulation of each enzyme in the pathway.
We are therefore not able to predict precisely and
reliably the effect of the insert on PHBV production.
GMO Bacteria
The utilization of GMOs causes debate in some countries.
That’s why we need to be sure of
the absence of genetic materials or residue of cells in the final solution of PHA.
We can be
sure by the pasteurization that no one cell can live after the treatment and during the
purification protocol, we can pass through two times or more to be sure of the purity of the
PHA extract.
This point must be added in the safety form in the case of further
experimentations.
Cost of the production
Nowadays, bioplastics are still underused by the high price of their production. 40%
(Albuquerque and Malafaia, 2018) of this price is due to the carbon source and another major
part of the final cost by the purification step.
We have solved the first problem and
decreased the cost of production of our PHA. However, the purification step remains and
can’t be bypassed.
It contributes, with multiple cycles of culture, to the cost of
inflammation.
A problem of biodegradability
As reported by JF Ghiglione, biodegradability depends on the condition in which bacteria
able to degrade plastics can live and proliferate.
In oceans, the final acceptor of all
garbage, the temperature, the diminution of oxygen, or other components decreases largely
the probability of degradation of these polymers.
For example, bacteria that can degrade
bioplastics polymers require, for the most part, high temperatures (superior to 30°C and can
reach 70°C).
A polymer considered biodegradable can remain undamaged if environmental
conditions do not allow bacteria to live and degrade it.
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