Using Biology To Downstream Process Biology

Is there a flywheel here?

Welcome to the January Feature at The Polymerist. I’m definitely getting outside of my comfort zone and I’m writing about fermentation and biology and downstream processing. I have some familiarity with the area, but by no means am I an expert. That’s why I spoke to the CEO and CTO of a start-up called Bioextrax who are using biology to make downstream processing of poly(hydroxyalkanoates) (PHAs) easier. I think the concept of using biology not only to make our target molecules, but to also purify our target molecules could be the great unlock we need bring in the biomanufacturing revolution that I’ve been told about for the last 20 years.

This issue and the next are sponsored by:

I think that biomanufacturing via fermentation with wild microbes, metabolically engineered microbes, and chemoenzymatic synthesis are going to be very useful tools in the future as chemical companies seek new ways to produce both classic and new chemical products. I think metabolic engineering and fermentation hold a lot of potential for chemicals that are complex, possess chirality, or functionalities difficult to achieve via traditional organic synthesis.

Complex multistep organic synthesis that possess low yields have been done in microbes. Maybe start here if you are interested and go here and here. I supply these references for those interested in diving into the details and really this is just the tip of the tip of the iceberg. There is an enormous amount of value in doing relatively low yield fermentations if very complex molecules can be achieved with greater efficiency than traditional synthetic organic chemistry. I’m not a medicinal chemist nor am I a true organic chemist. I’m just a polymer chemist and this post is really about polymers (I promise, I get there eventually).

If we want to produce a significant amount of chemicals that are cost competitive with traditional chemicals then current production costs will have to come down for fermentation based technologies. For a good overview of fermentation technologies check this out from Arye Lipman. There are a few areas that can be looked at to remove costs such as increasing production or reducing costs of feedstocks. 

What Is Downstream Processing?

One area to remove a significant amount of cost is simplifying the downstream processing (DSP) to isolate the desired product. DSP is what has to be done once the microbes are done fermenting. A great example downstream processing of fermentations that occurs at scale is exemplified in making bourbon.

When making bourbon you need  to isolate the ethanol and the flavorful aromatics from the mash of the fermentation. A popular route for isolation is through a beer still and then a standard distillation. A beer still uses gravity to filter out the solid biomass and water and it simultaneously uses heat to distill over the ethanol. Essentially, you drop the fermentation mash down a big distillation column and use gravity to filter out the heavy stuff and heat to distill over the volatile stuff (it’s not that easy, do not try it at home).

One difference between bourbon and chemical products is that the impurities from the fermentation that carry over in the distillation for bourbon is where a significant part of the value is captured and why it's kind of an art and a science. Even more value is created in the aging and the interaction of the spirits with new charred oak barrels where flavors get added to the product. That’s the romance of making bourbon.

Source

For chemicals, we typically want no impurities. The less impurities the less chances there are for unwanted side reactions or unpredictable outcomes. Impure chemicals means unpredictable outcomes or unwanted side reactions that reduce yield. This translates into a significant amount of risk for everyone. These are the challenges that fermenters of chemicals deal with, but with less historical knowledge on which they can stand. We’ve been fermenting and isolating ethanol for centuries. We have maybe been fermenting high value specialty and pharmaceutical grade chemicals for a few decades?

Backyard Compostable Polymers

One area for polymers that has held a significant amount of promise that is still unrealized is fermentation of poly(hydroxyalkanoates) or PHAs. We can think of PHAs as a way that microbes or plants store energy for consumption later, kind of like fat, but the kind that plants can use. If we look at the structure of a common PHA we have three classics in the figure below. The idea behind PHAs has always been one of combating plastic waste via polymers that give us similar properties to polyethylene, polystyrene, and polypropylene, but can be composted in your backyard. The dream has been around since the late 1980s.

By Berserker79 at the English-language Wikipedia, CC BY-SA 3.0,

I’m not here to really write about PHAs in depth. Some people think they are a viable solution to the plastic waste problem while others regard them as adding complexity to an already difficult to manage waste stream. The biggest feature of a PHA is that they have an ability to break down in backyard compost piles and degrade in marine environments. This is because PHAs are like food for certain microbes so when they are encountered in the environment they get eaten!

A Word From My Sponsor

Imagine going from lab scale to pilot scale and having your polymer work just as you thought it might. Then you go from pilot to commercial sized reactor and then things do not go as planned when analyzing it despite a smooth manufacturing trial. Now you have 16 tons or more of unusable product that you scaled up even though all of your analysis telling you the polymer is the same. It’s even worse when its your customer telling you it’s not the same. It might be time to get another opinion.

Reach out to Thomas Gungor at thomas.gungor@polyanalytik.com to get started!

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A Brief Summary of PHA Commercialization Attempts

Metabolix was the first company I learned about when it came to making plastics from fermentation, specifically with PHAs. Metabolix was founded in 1992 based on technology discovered at MIT. One issue around PHAs was getting the downstream processing figured out and having an extrudable and injection moldable resin. This was figured out eventually and Metabolix was ready to scale with a key partner that backed out at the last minute. A good article from Alexander Tullo in C&EN details how slow market adoption and a myriad of pivots to other products were the beginning of the end for Metabolix. They eventually went bankrupt. The shareholders eventually pivoted the remaining IP to become Yield10. 

I bring up Metabolix because Danimer Scientific is in a similar position now and we could be on the cusp of a PHA revolution in the next few years as capacity is built out for the fermentations. PLA or polylactide (also known as polylactic acid) was the first commercial scale biodegradable plastic, but it has been facing its own issues around compostability. PHAs have more properties that can be fine tuned through fermentation conditions when compared to PLA and in theory offer significantly easier routes to making very specific polymers for specific end uses with marginal increases in cost.

Last year, NatureWorks (largest producer of PLA in North America) and IMA Coffee entered into a partnership to develop compostable K-cups and recently Keurig was fined 3 million dollars for claiming their pods were recyclable. PHAs have been shown to have better compostability than PLA in different environments, but this has yet to be proven at scale in commercial applications. I suppose we will find out soon as Danimer is expanding their production capacity in Georgia to the tune of $700 million.

One area where I think PHAs have a real strength over PLA is that they have near infinite ability to be fine tuned on the metabolic level to yield very specific polymers for specific applications. Here is a good review article by Professor George Chen for those interested. This metabolic flexibility of PHAs is unique and we should be thinking about exploiting it for the future, but the problem of downstream processing still persists.

How do you selectively remove the biomass you don’t want and keep your polymer intact? Is there a beer still for PHAs?

Bioextrax

I met Mohammad Ibrahim when he came to the US to do a postdoc with Richard Gross when I was a graduate student. We worked together briefly on a bacterial cellulose biobased epoxy composite idea that got presented at an ACS conference,¹ but eventually I graduated and Mohammad went back to Sweden to found Bioextrax. I got to catch up with Mohammad and his co-founder Edvard Hall recently on what Bioextrax is doing and where they are headed.

Bioextrax has grown the last time I checked in with Mohammad. They have 15 employees now and are in licensing agreements with a few different customers for their downstream processing technologies. It’s amazing to me how far they have come since Mohammad first told me of the idea years ago, but what exactly does Bioextrax do?

A Microbe That Eats Other Microbes

Bioextrax is using a microbe to selectively degrade the cellular material that surrounds PHA granules after the fermentation has finished. The Bioextrax microbe isn’t genetically modified and it selectively degrades the non-PHA biomass so that it is easier to isolate the polymer. Traditional methods to isolate PHAs have been using solvents (e.g. chloroform) where solvent ratios could be 20:1 or using acetic acid to lyse the cells. 

No one in the chemical industry likes using solvents due to the following reasons:

1. Pose a safety risk with toxicity and flammability

2. Environmental risks in loss of containment or unpermitted release to atmosphere

3. Require energy to purify through distillation for recycling (if that capability exists)

4. Have to be disposed of as chemical waste when unable to recycle (costs money)

The more modern route these days is to use a weak acid to lyse the cells or use an enzymatic cocktail of proteases and lysozymes. The enzymatic route can be pricey for most PHA producers because these specific enzymes are expensive. It’s not like there are a lot of PHA producers out there clamoring for specific proteases and lysozymes that could yield a volume discount. 

Keeping costs low is important to be able to sell PHAs that compete with traditional petroleum based plastics. A great unlock around the enzymatic route would be to lower the cost of the enzymes needed by 10-100x. The acidic degradation route could yield high purity and high yield, but careful control of the conditions are important to minimize PHA degradation.

Edvard told me that the feedstock going into making the PHAs are important too and companies such as Full Cycle Bioplastics or Genecis who use waste feedstocks to make their PHAs are sensitive to how the downstream processing of their PHAs occurs. Acid degradation can also degrade the very polymer these companies are trying to isolate. If too much degradation occurs then this can lead to the PHA not having the correct materials properties needed to be useful. I couldn’t find a good paper on acid/base strength influences on PHA degradation, but here is a paper on glycerol based polyesters degradation. 

When it comes to degradable polymers the challenge isn’t making them degradable, but rather having them degrade when you want them to, and this typically occurs under very specific conditions. This means a long shelf life when humans are using them and ideally rapid degradation when we are finished with them. Polyesters are a class of polymer with amazing potential for a wide range of diverse end market applications and potential degradation triggers such as acids, bases, and enzymes are most common, at least for now. Having a PHA degrade too early defeats the purpose of the polymer.

The ideal place for a PHA to degrade is through biological degradation in a composting system. Bioextrax also has a microbe that can degrade PHAs back to their monomers completely so in the event PHAs can be recovered intact there is an easy route to turn them back into their monomers, which is an interesting route to the PHA monomers because PHA producing microbes use other carbon sources such as food waste, methane, sugar, or waste water as their feedstocks. 4-hydroxybutyrate doesn’t just hang out with sugar on the weekends.

Edvard is most excited about PHA use in personal care products such as skin creams and as a polymer to coat paper. Personal care products have the potential to accept the high initial costs of PHAs as fermentation capacity is expanded and could be an end market where the diverse functionality of PHAs could be fully realized. At this stage for PHAs I think showing profitability early despite high costs is the most challenging aspect of commercialization.

Coating paper with hydrophobic PHAs could be a real packaging solution to our current state. Right now, most people don’t know where to put a paper cup (either hot or cold). Should it be recycled with other paper, composted, or put in the trash? The answer is that a paper cup today should probably go into the trash because of the polymer coating (usually polyethylene, sometimes PLA) that keeps the paper from saturating with water and leaking everywhere. A PHA coated paper cup on the other hand could be composted.

Edvard and Mohammed have vision outside of PHAs too. Their next target is looking at how to degrade poultry feathers to make Keratin microfibers. Here is a free and easy to read review on the uses of Keratin. Here is an NPR story on chicken feathers that featured Professor Richard Wool. Professor Richard Wool was a pillar of the green chemistry and materials community and he sadly passed away in 2015. 

Right now, the world produces about 6 billion pounds of poultry feathers that are considered useless. Chicken feathers typically go to landfills where they add no value, but unlocking a use for these feathers could be another way to generate useful materials for very little raw material costs. 

Low raw material input costs, turning waste streams into high value materials, and using biology as a way to not use external energy for the transformation sounds incredibly lucrative. The trick is making a material that people want at a cost they are willing to pay. It’s easy to see the applications, but it’s hard to make a commercially competitive product.

Using biology to do the downstream processing instead of relying on traditional chemical engineering techniques seems like the final unlock that we need to usher in the biomanufacturing revolution that I’ve been hearing about since I started graduate school 10 years ago. I think Bioextrax could be the unlock that many PHA producers are looking for and there once they have isolated their PHA from the biomass they are left with a bunch of non-food protein. 

Anyone know what you can do with a bunch of cellular protein biomass?

Bioextrax is a great example of what else can be done with biology outside of the traditional fermentation to product concept. If you are interested in investing or contacting Bioextrax to trial their technology I recommend reaching out to Edvard directly at edh@bioextrax.com. 

Tony

1. Biosynthesis of ultra thin nano fibrous bacterial cellulose (BC) film for new applications

Fei Liu, Mohammad Ibrahim, Anthony Maiorana, Michael McMaster, Lu Li, Shekar Mekala, Kyle Peters, Cameron Kee, Kenneth Singer, Nikhil Koratkar, Richard Gross, ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY, 2017