For the first time, researchers have managed to produce three products in three separate phase states in a single process carried out by a microbe.

The work is a joint partnership between the Chen Lab, a biodegradable-plastic powerhouse in Tsinghua, and the Scrutton lab, a propane/biofuel powerhouse lab in Manchester, formed in order to sustainably produce three valuable products in a single fermentation process.


The industrial plant in China

The procedure, which produced gas in the form of propane, liquid in the form of mandelate, and solid in the form of biodegradable plastic (PHA), is outlined in a study in Microbial Biotechnology, an Applied Microbiology International publication.

Helen Park, who worked on the research at the Scrutton lab and is now working in the Chen lab, completing the process that will scale the project to industry, said the aim was to make three products at once without decreasing the titre of any one product.

Sustainable transition

“We are motivated to speed the transition from fossil-fuel derived plastics, fuels, and materials to a sustainable future. Metabolic engineering is key to replace the harmful effects of fossil fuels because we can make the same compounds as in the oil and gas industry, but sustainably through the genetic engineering of cells,” she explains. 

“In this work, we make three sustainable products that are valuable and in high demand. Importantly, by producing all three products at once, we can drastically cut down the cost of fermentation – otherwise we would need to run separate processes for all three, which is time consuming and expensive. 

“Since the major holdback to scale-up of bioprocesses is cost, we are motivated in this work to decrease costs through three-product production. We picked three compounds that are both valuable, but also separate into three phases naturally in the fermentation. 

“By having a solid, liquid, and gas, we can easily separate the compounds in the fermentation and therefore again cut down costly purification. 

“Finally, we picked the industrially important extremophile Halomonas, which is a bacterium isolated from a salt lake in China that can withstand huge changes in salt, pH, temperature and is resilient to many toxins. This bacterium can, importantly, grow in nonsterile conditions without contamination – which is very rare to find in industry. Avoiding sterilization - which tends to be the biggest cost in industry - is therefore achieved in our work.”

‘Drop-in’ biofuel

Propane was chosen as an ideal  ‘drop-in’ biofuel that that can be liquified and added into cars, planes, etc along with gas, which makes it of high value with a market size that is growing every year. 

“For propane, we used a light-inducible enzyme (CvFAP) that is normally found in fluorescent organisms. This cool enzyme was engineered so that when it is hit with blue light, it will produce propane,” Park says. 

“In our work, we integrate the enzyme into our host organism, Halomonas, and activate it at different intervals to produce and collect propane. The production of propane is timed carefully to not interfere with the other two products, and we performed many series of fermentations to get the growth and ‘light-on, light-off’ timing perfectly.”

Plastic production

Meanwhile, PHA, a biodegradable plastic that is poised to replace fossil-fuel derived plastics, is made naturally by Halomonas when the fermentation conditions are right.

“We need to tune the amount of glucose, nitrogen, and other media additions just right so that we can ‘harvest’ the cells for their PHA at regular intervals,” Park says. 

“We took design of experiment statistical testing in order to optimize conditions for PHA and for our third component, mandelate. We found we could ‘spike’ the glucose for PHA at the right time before harvesting cells, then we could use all old media - no need for any waste in the process- and reinoculate with fresh cells, making a semi-continuous process and saving costs.

As for the mandelate and hydroxymandelate, these two compounds are of high value, and were added to the fermentation process to increase the value of the final products, as both can be used in materials and in the drug industry. 

“We envision this process could also produce other third products besides mandelate, but we chose mandelate for proof-of-concept, because it had never been made before in Halomonas and indeed is rare to find produced sustainably yet - most mandelate is made in the chemical industry,” Park says. 

“We optimised the mandelate titre by adjusting the media feed, by adjusting the induction time of the enzyme (cell growth), and a variety of other factors. The mandelate is soluble in water and therefore separatable from the other two products.”

Three products

The team succeeded in producing all three products and found the propane titre was higher than ever previously reported in a biological process.

The mandelate titre is the first successful production in an extremophile bacterium, and the PHA titre is similar to prior results - nearly 88% of the cell’s final volume is full of plastic. This success means that indeed three products can be made in one organism without damaging the titre of any one, which is going to be a big deal in industry in terms of cutting costs,” Park says.

The next step is to test new three-product systems and scale to industrial levels.

“The work was caried out in a small bioreactor, and we have previously seen that at scale the titre will actually increase, because cell density can increase by a factor of 10-100!” Park says. 

“In Manchester, the Scrutton group, through FBRH, is in the process of building a propane-production plant, in order to test this process at scale. 

“Meanwhile, the Chen lab in China - I am now here, completing the project - is working directly with industrial partners to scale the PHA, mandelate, and other compounds to 225,000 L bioreactors. The hope is that in the coming years these products can be made sustainably, and become cost competitive with fossil fuels.”

’Co-production of biofuel, bioplastics and biochemicals during extended fermentation of Halomonas bluephagenesis’ is published in Microbial Biotechnology.