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


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 - 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.


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 –
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. 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.

Step 4: Transformation in BL21 E. Coli.
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).


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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