Introduction

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The implementation of PETal has been developed with the project's core values - quality, sustainability, and innovation in mind. PETal proposes a novel way of synthesising sandalwood oil using biotechnology, using PET plastic monomers Terephthalic Acid (TPA) and Ethylene Glycol (EG) as feedstock.

Framing the Problem

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Sandalwood Essential Oil (SEO) is one of the most expensive and sought-after plant essential oils. It is used in fragrances, soaps, ittars, and incense sticks. Santalum album, a sandalwood species native to southern India, is known for its high santalol content, the molecule that grants sandalwood oil its rare properties. The oil is extracted from the heartwood of the tree.
Like other essential oils, SEO is found in deficient concentrations in the tree - 1000 hectares of mature (20-30 years old) sandalwood trees produce 8000 ADTH (Annual Dry Tonnes per Hectare), which can only yield up to 200 tonnes of oil, i.e. only 2.5% ^[1]; therefore, many trees need to be harvested to extract a substantial amount of essential oil. Consumerism has led to a surge in demand for cosmetic products, and sandalwood’s rare cosmeceutical properties expose it to overexploitation and increase its susceptibility to illegal smuggling.

Efforts have been made to tackle this issue; let’s better understand it by studying the life cycle of an essential oil.

Fig. 1: Life-cycle of an essential oil

Fig. 2: Chemical Synthesis of an essential oil

Fig. 3: Natural Synthesis of an essential oil

Fig. 4: Biotechnological Synthesis of an essential oil

After studying the three production methods of essential oils, it is evident that the benefits of using biotechnological methods outweigh the benefits of the other two alternatives. Synthetic biological systems are extensively used to produce macromolecules of interest using microbial systems. Therefore, we decided to take advantage of the potential of biotechnology to synthesise sandalwood oil.

However, we wanted the process to be completely sustainable. While brainstorming to find the feedstock for our bacterial system, we stumbled upon the groundbreaking research done by Dr. Joanna C Sadler ^[2], who managed to convert PET plastic to Vanillin in 2021.
Dr Sadler (read more about our conversation with her here) is very interested in upcycling plastic and exploring more sustainable alternatives for adding value to plastic. Our conversation with her was pivotal in our project. We decided to explore PET plastic as a feedstock further.

Plastics are synthetic materials made up of polymers derived from fossil fuels. They’re a chain of carbons that do not directly participate in the carbon cycle and act as significant land and water pollutants—using them as feedstock in bioprocessing poses an opportunity to release these carbons into the environment as feedstock for bacteria to carry out metabolic processes while also tackling plastic pollution.

Converting plastics to bioplastics or textiles is a standard solution to the plastic problem. However, that doesn’t entirely solve it. While repurposing plastics to bioplastics requires less energy than synthesising virgin plastic, bioplastics and synthetic fibres aren’t necessarily environmentally friendly ^[3],[4],[5].

Fig. 5: Comparative analysis of different ways of upcycling plastic

Unlike the other alternatives where plastic is being converted to more plastic despite the efforts of upcycling it, upcycling plastic into value-added products like essential oils removes it from the equation entirely, uses degraded plastic, changes its chemical composition and upcycles the monomers instead. We believe this approach can offer a long-term and sustainable solution to plastic pollution ^[6],[7].

The conversion of PET plastic to Sandalwood Essential Oil could solve both problems - the gap between supply and demand for sandalwood oil due to its overexploitation (a local problem) and the ever-rising problem of plastic pollution (a global problem).

We then formulated PETal - tackling a local problem, using a global problem as a solution - and synthetic biology makes it possible.

Developing a Modular Working Model

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Within the scope of the timeline of the competition, we developed our project intending to make P. putida KT2440 optimised strain - P. putida TA7EG to assimilate PET monomers Terephthalic Acid (TPA) and Ethylene Glycol (EG) and synthesise santalol. We developed this system by codon-optimising santalol synthesising genes found in the Santalum album plant to be expressed in a prokaryotic system, like P. putida, by integrating it in plasmids specific to the bacteria (read more about this on the Project Design page).
Towards the end of iGEM, we were able to successfully achieve the first-ever synthesis of santalene in Pseudomonas putida with the SaSSy_FPPS plasmids in the pSEVA-631 backbone - a plasmid designed by our very talented dry lab team.

Motivated by our achievement, we aim to extend the project's potential beyond what has been achieved thus far and complete our proof of concept to take PETal from the lab bench to the industrial level. The broader goal of our project is to not only finish our proof-of-concept and optimise our current strain for industrial applications but also develop a modular working model that can be used for synthesising a variety of plant terpenoids. Further, we plan to consider the carbon economy of our model and address the biosafety concerns associated with the project. Towards the end, we’ve devised a model of a synthesis unit, which will contain a unit for processing plastic waste, a bioreactor, and necessary means for the downstream processing of the synthesised santalol.

Optimising P. putida TA7EG for Industrial Scale-Up

Currently, the santalol synthesising enzymes Santalol Synthase (SaSSy), Farnesyl Pyrophosphate Synthase (FPPS), Cytochrome P450 Monooxygenase (CYP450) and Cytochrome P450 Reductase (CPR1) have been transformed into the bacteria using plasmids. To carry out differential heterologous gene expression in bacteria to achieve chemical conversions, plasmids are a convenient choice, as the plasmid strains can be modified to tune gene expression and high titers ^[8]. However, challenges are associated with using our current strain on an industrial scale.
Challenges:

1. Inducible promoters are both metabolically and economically expensive at an industrial scale. Expression of plasmids depends upon inducible promoters, which requires the bacterial density to be monitored, hindering the automatisation of the induction of expression. Apart from this, the induction of plasmids during exponential growth may increase the metabolic burden on the bacteria ^[9].
2. Industrial production of chemicals via bio-based routes often faces limitations due to plasmid instability. Since bacterial replication is exponential, the segregation of plasmids may not be uniform amongst all the progeny. This can result in difficulties in bioreactors due to the presence of productive cells (cells containing plasmids) and non-productive cells (cells not containing plasmids)^[10],[11]. Environmental stresses and lack of selective pressure can result in plasmid instability.
3. Achieving high titers of the final product without plasmids. Plasmids have the ability to regulate gene expression by manipulating their copy numbers within the bacterial cell. However, by removing plasmids from the equation of heterologous gene expression, over-expression of genes can potentially lead to a bottleneck in product synthesis.

Genomically Integrating Recombinant Genes into the Bacterial Chromosome

To learn more about the industrial production of essential oils produced using synthetic biology, we spoke to Dr. Joanna C Sadler . Addressing our concerns, she recommended that we genomically integrate the genes of our interest into the bacteria's chromosome. This way, we could bypass the process of inducing the plasmid to activate the synthesis of genes responsible for santalol synthesis.

Before genomically integrating a set of genes into the chromosome, it is essential to answer a set of questions - What genes are to be integrated? Where do we integrate them? And how do we integrate them?

We intend to integrate the Santalol Synthase (SaSSy), Farnesyl Pyrophosphate Synthase (FPPS), Cytochrome P450 Monooxygenase (CYP450) and Cytochrome P450 Reductase (CPR1) genes into the bacterial chromosome. Their eukaryotic origin could make it difficult to directly express these genes into the bacterial genome due to dissimilarities in codon usage. To overcome this, we’ll codon-optimise these genes to be expressed in a prokaryotic system ^[12].

The site of integration of the genes plays the most significant role in the regulation of the expression of genes. Recent studies on Pseudomonas putida have seen increased expression levels across different cultivation conditions in the P. putida genome, and these sites are called “landing pads” ^[11]. Landing pads were characterised using RNA-seq data, and sites showing low variability across the genome were chosen. Further, it was demonstrated that promoter-independent gene expression variations are controlled by their integration in the genomic “hot” or “cold” spots. Therefore, the genomic context of integration has proven to be an essential toolbox for gene integration in Pseudomonas putida. Another set of studies has shown the pipeline for screening for integration sites computationally by selecting sites non-proximal to essential genes, non-operon interfering and using downstream genes in antiparallel orientation ^[13]. The effect of gene integration on growth and expression was measured, and 5 out of 7 integration sites had no adverse impact on the development of the bacteria. Differential expression was attributed to the change in proximity of the genes to the Ori region of the chromosome. The expression of heterologous genes is tuned according to the purpose of expression since the locus chosen affects protein expression and cell growth. Therefore, we intend to integrate the SaSSy, FPPS, CYP450 and CPR1 genes after analysing the RNA-seq data of Pseudomonas putida TA7EG growing on terephthalic acid and ethylene glycol - in the sites that show maximum expression and minimum adverse effects on the growth of the bacteria for industrial scale-up.

Finally, genetic tool-kits have been developed for synthetic biology in Pseudomonas. Gene integration by homologous recombination proved inefficient ^[14], leading to the development of other, more efficient methodologies. The TREX (transfer and expression) system of gene integration allows the random integration of genes across the genome using transposons where their expression is mediated by a T7-like RNA polymerase, a system previously used in Pseudomonas putida to achieve lycopene production ^{[15],[16],[17]}. While T7 RNA polymerase-mediated expression of genes has shown an increased level of expression when compared to the traditional systems of expression in P. putida ^[18], random integration of genes into the genome may not be ideal considering industrial applications of the strain. We hope that screening the genome for loci showing increased gene expression regardless of the external environment may help our cause. Further, for site-specific integration of genes, well-established synthetic biology tools like CRISPR-Cas9 are easy and reliable.

A similar strategy can also be employed to synthesise other terpenoids.

Implementing PETal and Scaling-Up in the Real World

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Introduction

To give a realistic picture of how PETal will be implemented in the real world, we devised a plan of a bioprocess synthesis unit that would take in PET as feedstock and, with continuous processing at each step of the unit, achieve the conversion of santalol.

Fig. 6: Graphical representation of the synthesis unit

This synthesis unit will contain:

1. A waste collection facility
2. A waste segregation facility
3. A facility for carrying out enzymatic hydrolysis of PET plastic
4. A bioreactor
5. Downstream processing facilities - cell lysis unit, HPLC and GCMS

In this segment, we have proposed the development of this synthesis unit as well as exploring the possibilities of taking our project further by exploring different feedstocks, documenting the catalytic activity of various naturally-occurring PET degrading enzymes which can be used by future teams working on PET plastic, developed a bioreactor for santalol production and a downstream processing facility. Later, we studied the governmental approvals required to assess the safety of our product and its launch into the market. To navigate through the process, we have divided the synthesis unit into Upstream Processing, Production Process and Downstream Processing.

Fig. 7: Upstream processing, Production process & Downstream processing

Upstream Processing

Upstream processing involves the first stage of bioprocess development, which includes the preparation of microorganisms or subjecting them to manipulation before the production process begins. During upstream processing, the culture media, temperature, pH, and oxygen conditions are optimised to best suit the requirements of the bacterial culture, which is to maintain biomass and synthesise the desired product. Efficient upstream processing prior to production promises better quality and yield of the final product ^[19].

1. Obtaining Feedstock
   
   The plastics found in the environment, such as Polyethylene Terephthalate (PET), Polyethylene (PE), Polystyrene (PS) and Polyvinyl Chloride (PVC), are made of synthetic polymers derived from petroleum. While PET is a heavily recycled type of plastic, its biodegradability was established only in 2016. Yoshida et al. 2016 ^[20] discovered a novel bacterium, Ideonella sakaiensis 201-F6, which could use PET plastic as its sole carbon source to fulfil its energy requirements. The bacteria secreted a hydrolytic enzyme called PETase, the characterisation of which led scientists to wonder whether other similar bacterial species and hydrolytic enzymes were present in nature.
   The biodegradable nature of PET is owed to the heterogeneous (C-H) bonds keeping the two monomers together.
   
   
   

   Fig. 8: Comparison between PET plastic and Polyolefins

   Plastics such as Polyethylene (PE) and Polystyrene (PS) are called polyolefins, i.e. their repeating units or monomers are alkenes. The C-C backbone of these plastics makes them resistant to biodegradation by microorganisms ^[21]. While scientists continuously look for microorganisms that can effectively degrade homogenous plastics, the studies have shown sub-par results with meagre degradation rates ^[22],[23].
   Fascinatingly, owing to its robust nature, Pseudomonas putida growing in textile effluent drainage showed intermediate levels of biodegradability for Polyethylene ^[24], proving that Pseudomonas can be trained to degrade PE efficiently through laboratory evolution assays and can be transformed with plasmids to synthesise essential oils. However, any such data for other plastics having C-C backbones remains unknown.
   
   Under the scope of the competition, we’ve chosen PET plastic as the feedstock for our bacteria.

2. Segregation of PET from Plastic Waste
   
   Post-consumer plastic consists of items of various degrees of heterogeneity and plastics comprised of a variety of polymers (PE, PS, PET, PVC, etc.); therefore, the next step after choosing plastic as feedstock is to decide how we’re going to segregate plastic, especially PET plastic from general waste since all post-consumer plastic generally looks the same. The most common method of sorting plastic from general waste is mechanical sorting, where objects and items made of plastics are picked up and segregated by hand. Not only is this process tedious, but it is also highly inefficient since not all plastic waste looks “plastic-y” ^[25]. To tackle the problem of ineffective sorting, scientists have devised several ways of automating the sorting process, mainly by using two approaches: sorting using sensors ^[26] or sorting using Artificial Intelligence (AI) learning models.
   The optical separation of plastics is done by using IR (infrared) or NIR (near infrared) lasers, while high-density polymers (HDPE and PET) could be separated using colour as well. Factors such as density, chemical composition, and colour of the plastic can help detect the plastic type by disrupting its spectra ^[27].Camera systems trained using machine-learning algorithms, combining training models with optical detection of plastic waste, are a more recent and efficient way to segregate plastic that has emerged. ^[28]
   After segregation, the waste must be cleaned and sterilised before processing. This can be done by washing with water or caustic soda or subjecting the waste to a steriliser.


3. Extraction of PET Monomers
   
   After the PET feedstock has been segregated and cleaned successfully, processing and monomerisation of the plastic comes next. Although chemical hydrolysis is the most efficient way to depolymerise plastic to its monomers, with recent discoveries in methods of plastic depolymerisation by microorganisms (bacteria and fungi) and macroorganisms (insects and worms), biodegradation is now being looked at as an alternative for industrial applications. As mentioned in this page before, the discovery of the PETase enzyme intrinsic to the bacteria Ideonenlla sakaiensis was a pivotal stage in the development of sustainable methods of plastic recycling, and moving forward, scientists discovered a variety of enzymes found across species that could degrade PET plastic. Along with this, efforts were made to optimise the current enzyme by using ML and DL learning models.
   
   The following table compares the Km, Kcat, rate of depolymerisation and thermostability of wild-type and optimised PET degrading enzymes isolated across bacterial species.
   
   

   Enzyme Name	Km value	Kcat value	Rate of Depolymerisation	Thermostability
   WT-PETase	370 mM	1.5 s⁻¹	10% after 24h	Highest enzymatic activity at 35 degrees Celsius
   Fast-PETase	0.60 mM	90 s⁻¹	98.2% in 8h	Tm at 63.3 degrees Celsius
   Thermo-PETase	0.12 mM	57.62 s⁻¹	14 fold as compared to WT-PETase [29]	Increased enzymatic activity up to 40 degrees Celsius [29]
   HotPETase	6.07 mM	82.5 s⁻¹	12% conversion after nine h	Tm at 80.5 degrees Celsius [30]
   WT LC Cutinase	0.50 mM	12.7 s⁻¹	93.2 mgTAeq. h−1 mgenzyme−1 at 65 °C	Enzymatic activity vanishes six-fold from 70 degrees Celsius to 80 degrees Celcius
   ICCG (Optimised LCC)	0.17 mM	36.5 s⁻¹	97.3% conversion	Tm at 91.7 degrees Celsius [30]

   
   It is to be noted that a low Km value signifies a high affinity of the enzyme for its substrate, allowing an enzyme with a low Kcat value to operate at a constant concentration at physiological conditions with little to no fluctuations in its activity. On the contrary, a low Kcat value is directly correlated with lower turnover rates.
   
   From this table, we can see that ICCG (optimised LCC) has the second-lowest Km value after Thermo PETase and has an average Kcat value. To better understand the activity of an enzyme, we typically look at the KcatKm value of the enzyme, which for Thermo-PETase is ~480 s-1M-1, while for ICCG (optimised LCC), it is ~280 s-1M-1. Despite the significant difference in the Kcat and Km values ratio between these two enzymes, ICCG (optimised LCC) emerges as a better industrial alternative, which can be owed to better thermostability ^[30]. Following the nearly complete depolymerisation of PET by ICCG (optimised LCC), we can conclude that enzymatic hydrolysis of PET is possible using bacterial PET degrading enzymes.
   
   With the enzyme decided, we will now proceed to the process of depolymerisation of PET plastic waste. Once sterilised, the PET waste can be subjected to mechanical milling, i.e. shredding PET to fine particles - to increase the surface area for the enzyme to hydrolyse the bonds and convert PET to TPA and EG. The temperature while depolymerising is taking place should be kept at temperatures close to the glass-transition-temperature of the PET polymer ^[30].
   
   The Terephthalic Acid (TPA) and Ethylene Glycol (EG) obtained after depolymerisation are filtered and used as media for the growing chassis, Pseudomonas putida.

Production Process

The production process of a bioprocessing unit involves carrying out the large-scale synthesis of the desired product in a bioreactor. Here, we have laid down a rough idea of what the bioreactor responsible for synthesising santalol will be like.
Bioreactor Design
To design a bioreactor for our project, we sought advice from professionals. To begin with, we had discussions with Dr Joanna C Sadler from the University of Edinburgh, who had previously worked on synthesising Vanillin from PET plastic (read more about our interaction here).

• She highlighted how the conditions exposed to the cells in a shaker flask and a bioreactor are different since the density of the cells and maintaining optimal growth conditions at a large scale come into play.
• She advised us to look into strain optimisation for better yield from our bacteria at an industrial scale.

Further, we spoke to Dr. G Suraishkumar from IIT Madras, who specialises in bioreactor design. (read more about our interaction here)

• After explaining our project design, he suggested that we stick to a simple stirred-tank type bioreactor where we mimic the conditions of the shaker-incubators we had been using thus far for growing our bacteria in the lab.
• He mentioned that since we use liquid media like TPA and EG, it would be easier to scale up production and manage the nutrient conditions.
• Since our final product, santalol, was to be extracted after lysing the bacteria, he suggested using a fed-batch type fermenter to grow our bacteria and achieve the requirement of higher cell concentrations.

By incorporating these suggestions into our working model, we devised an airlift-type bioreactor model to maximise the production of santalol. We chose airlift-type bioreactors over the conventional choice of stirred-tank reactors (STR) because of their ease of scale-up compared to the latter. STRs gradually lose the mixing quality as the bioreactor size increases. The simplicity in design, easy installation, lower power consumption and the ability to retain the mixing quality despite variations in size made airlift-type bioreactors an ideal choice for us ^[31],[32].

Features of the bioreactor:

1. Material of the bioreactor: The material for the bioreactor is chosen considering its resistance to chemical exposure, ability to withstand sterilisation, mechanical properties and cost. Our bioreactor vessel will be made of stainless steel, which is commonly used in bioreactor design due to its corrosion resistance and chemical exposure.
2. Media input: The media will be M9 minimal media containing TPA and EG, which will be supplied to the vessel via a shaft. Additionally, we plan to introduce alternative carbon sources like glucose from pre-treated agriculture and food wastes or sucrose derived from an autotroph in an effort to make our process carbon-neutral.
3. Sensors, Controls and Parts: Sensors for pH, temperature, oxygen levels and DO will be installed in the vessel. Further, vessels will also be used to detect the levels of TPA and EG in the culture. The media supply into the vessel will vary depending on the concentration of TPA and EG at any given time. A sensor-mediated approach to cycling media will keep wastage at a minimum. The bioreactor model will also consist of a baffle - to break vortex formation within the vessel and a jacket - to minimise heat transfer and maintain a constant temperature inside the vessel.
4. Functioning: The bioreactor vessel consists of two parts - a riser and a downcomer. Inside the vessel, mixing takes place with the help of air/gas. The air/gas is fed to the bioreactor via a gas flow inlet at the bottom of the vessel via a sparger in the riser part of the vessel. An airlift-type bioreactor differs from a STR since there’s no mechanical mixing of components inside the vessel. Hence, risks caused by shear stress to the bacterial cells are decreased at lower costs ^[33],[34].

Constraints in the bioreactor:

1. Oxygen availability: Pseudomonas putida is an aerobic bacteria. In large-scale bioreactors, oxygen becomes a limiting factor due to solubility issues of oxygen and inefficient mass transfer rates in the bioreactor. Accumulation of CO2 in the bioreactor can affect the availability of oxygen.
2. Temperature control: Most aerobic reactions are exothermic, so maintaining a constant temperature of 30-37°C for growing P. putida throughout the process becomes essential. RTD or Remote Temperature Detectors can help maintain constant temperatures ^[35]. Necessary cooling systems should be effectively integrated into the vessel system. Temperature Control Units (TCUs) are installed into the vessel, offering heating and cooling capabilities for managing endothermic and exothermic reactions.TCUs can be connected to the bioreactor jacket ^[36].
3. pH control: pH conditions of the media change as the bacteria grows and releases waste products. To maintain optimal pH levels between 7 and 7.5, practical strategies to control the pH levels should be employed, such as pH control systems. Real-time pH monitoring systems using proportional-integral-derivative (PID) controls. The addition of acids or bases as required to calibrate the pH takes place, and in some cases, CO2 levels can be adjusted to normalise the pH values inside the vessel ^[37].
4. Nutrient availability: In continuous fed-batch reactors, managing optimal nutrient conditions is essential to avoid overfeeding or underfeeding the bacteria. Inadequate levels of nutrients in the media can adversely affect production. Therefore, sensors to measure levels of trace metals, TPA, EG, and growth media will be installed to monitor the nutrient media continuously. Predictive models like Model Predictive Control enable the automation of nutrient management.
5. Foaming and frothing: Continuous agitation in high-density cell cultures can result in the liquid culture developing froth and becoming viscous, leading to loss of culture and sub-optimal functioning of the bioreactor. Anti-foaming agents can be added to the culture media to avoid such circumstances.

We plan to use Model Predictive Control (MPC) to overcome the challenges presented by the variability in culture conditions inside a bioreactor vessel. MPC is an advanced control strategy that can be used to predict the future of the bioreactor conditions. MPC has developed systems that can operate independently without expert interventions for a long time ^[38]. Experimental data is obtained and fed to computational models for running simulations. MPC systems help run reliable simulations for non-linear systems where straightforward predictive models often fail. MPC models have demonstrated the ability to predict future viable cell concentrations using present data and can further predict feed and bleed flow rates. The predictive capabilities of MPC models reduced variability in biomass over an extended period of time and helped improve product and yield efficiency ^[39]. MPC can be integrated with temperature and pH control systems along with feedstock control to give situational predictions more accurately ^[40].

We’ll use a bench-top bioreactor to precisely assess the media, initial pH, oxygen, temperature and agitation speed values ^[41] . The obtained values can be tuned for larger bioreactors with 800-1000L working media.

Fig. 9: A typical air-lift bioreactor

Downstream Processing

Isolation, lysis, extraction, and quality checks constitute an industrial bioprocess's downstream processing and the last stage. The downstream process is the most important step in this series, which determines the product's quality, purity and yield. The ease of implementing the downstream process determines the overall simplicity of the production process, i.e., in cases where downstream processing is tedious and time-consuming, the bioprocess as a whole becomes tedious, and the vice versa holds true as well.
For the extraction of santalol, we have laid down the steps required for the downstream process.

1. Extraction of Santalol
   Assuming our Pseudomonas putida is a whole-cell microbial factory synthesising santalol, it is necessary to remember that santalol is an intracellularly synthesised product in P. putida i.e., to get santalol out of the bacteria, we would need to lyse the cell. The requirement of cell lysis after each batch to extract santalol is significantly more expensive than the bacteria releasing the santalol out of it since cell-lysis is more resource intensive and requires an extra cost of installing necessary equipment. Lysis can be carried out mechanically or using high levels of pressure.
   
   Our experimental data specifies that the bacteria synthesise santalol after achieving an OD of 600nm. Therefore, the first step of downstream processing - extraction of santalol, begins after the bacteria has achieved 600nm optical density.
   The culture is first taken from the bioreactor and sent to a centrifuge where the high rpm helps separate the culture from the bacterial cells. Once isolated, the bacterial cells are lysed in a media containing hexane, allowing the two organic compounds (santalol and hexane) to mix. Separating the bacteria from the culture can be made more efficient using biosensor-based screening methods to identify the strain with a high throughput value. Such a system has already been achieved for the biotechnological synthesis of vanillin ^[42], and we aim to adapt to a similar system for santalol synthesis.
   
   Since hexane and santalol have different boiling points, the mixture of santalol and hexane can be subjected to pervaporation^[43], where hexane evaporates, leaving behind santalol. Again, a similar methodology has been applied to the industrial biosynthesis of vanillin ^[44].
   Further, santalol can be quantified using HPLC and GCMS facilities.
   


2. Purification of Santalol
   The santalol obtained can be purified using more sensitive centrifugation methods to separate any solid contaminants, lingering growth media or debris from the liquid. A major concern of the production of santalol from PET plastic is the possibility of contamination by microplastics. To tackle this problem, we’re taking help from the iGEM REC Chennai team PlastiCops, where they’re synthesising peptides with specific affinity tags for various types of plastics, one of which is PET. Using their high-affinity peptides inside the surface of the santalol purification facility could potentially avert the risks of microplastics by capturing them and not allowing them to seep into the santalol.


3. Quality Control and Safety Considerations
   The purity of the extracted santalol will be checked against naturally occurring sandalwood oil. The first batch of bioreactor-produced sandalwood oil will be subjected to pre-clinical and clinical trials to identify and avert potential risks like allergens and toxins and fortify our product claims for a smoother marketing campaign. A variety of assays, including a microbiological assay to detect contaminants, stability testing to test the shelf life and chemical properties of the product, toxicological testing, skin-irritation and sensitivity testing, and safety testing for detecting the presence of heavy metals will be performed as a part of the pre-clinical and clinical trials^[45],[46].
   Safety considerations while performing the experiments can be found on the Project Safety page.


4. Governmental Approvals
   The Cosmetics Rules, 2020 defines the word “cosmetics” as an “article that intends to be poured sprinkled rubbed or sprayed or introduced into or otherwise applied to any human body or any part thereof for cleansing, beautifying, promoting attractiveness or altering the appearance and also includes an article intended to use as a component of cosmetic”. The santalol synthesised at PETal is not a complete cosmetic product; it is a cosmetic ingredient that can be used to develop cosmetic products. In India, the main body looking after approvals and regulations related to cosmetic ingredients and cosmetic products is the CDSCO (Central Drugs Standard Control Organisation). Further, the Bureau for Indian Standards (BIS) verifies whether the product falls under the GRAS (Generally Recognized as Safe) category. Here is a comprehensive list of GRAS cosmetic raw materials issues by BIS.
   Successful manufacturing of santalol by PETal will be supplemented with exhaustive pre-clinical and clinical trials and approvals by the CDSCO and BIS to ensure complete user safety and a strong brand identity.


5. Entering the Market
   The overall development of PETal as a cosmetic raw material would have been incomplete had we not considered our product's socio-economic implications and marketing. We believe that a product's true quality is reflected in its marketability. To find out more about how we analysed the overall market, understood our target market segments, conducted user and stakeholder analysis, risk analysis and established a future financial and operational timeline along with predicting the direct and indirect impacts of the project, head over to the Entrepreneurship page.