On Fermentation

“The role of the infinitely small in nature is infinitely large.”
Louis Pasteur

Fermentation is the first example of biotechnology in human history. While spontaneous fermentation predates the human species, the earliest human efforts at fermentation date from the Neolithic period (7,000 to 1,700 BCE). Texts and pottery dating to 7,000 BCE were discovered in China, documenting the conservation via fermentation of the milk of sheep, goats, camels, and cattle. Wine-making dates to around 6,000 BCE in the Caucasus region of Eurasia, and there is also evidence of fermented beverages being consumed in Babylon around 3,000 BCE.

It wasn’t until the 18th century, however, that people began to understand the fermentation process. In another technology first, the ability to grind glass into early microscopes by Antoni van Leeuwenhoek led to the description of yeast as "the ferment put into drink to make it work; and into bread to lighten and swell it.”

For almost two hundred years after van Leeuwenhoek’s work, yeast was considered merely a chemical agent helping to initiate fermentation, with great impact on the economy where beer and wine had critical roles. In the 1850s and 1860s, several French scientists finally showed that yeast was a living organism. Louis Pasteur demonstrated experimentally that fermented beverages result from the action of living yeast transforming glucose into ethanol. By helping solve an issue with a beer batch that smelled like bad milk, he also discovered there were several types of fermentations (e.g., alcoholic and lactic acid) driven by two different microbes (yeast and bacteria). His research led to, among other things, the publication of several books on the brewing of beers, the transformative process of pasteurization to preserve foods, vaccines for rabies and anthrax, and today’s broadest definition of fermentation: the microbial transformation of a sugary substrate.

Later in the 19th century, a German chemist named Eduard Buchner detailed the cellular machinery necessary for the fermentation of sugars by showing it could occur in yeast extracts free of cells. The in vitro study of fermentation and the description of the enzymes allowing it led him to be awarded the Nobel Prize in Chemistry in 1907 and opened the way to a new field of research: biochemistry.

Since the earliest days of human’s exploiting fermentation, introducing random mutations followed by strain selection were common practices to increase alcohol and fermented food production.  The phenomenal progress in molecular biology — the discovery of DNA structure by Watson, Crick, and Franklin in the 1950s, the development of new genetic engineering tools in the 1990s, and later, their generalization with tools such as the CRISPR-Cas system — led to the ability to manipulate cells specifically, and expanded the ability of scientists to engineer particular outcomes for both what and how much could be produced by fermentation.

The term “metabolic engineering” was first used in the 1990s to describe the practice of optimizing genetic and regulatory processes within cells to increase the cell's production of a certain substance. Scientists calculated a yield of useful products by following every atom’s metabolic journey from the substrate to products and byproducts. They identified parts of the network that constrain the production of these products and sought to modify them. Genetic engineering techniques were then used to modify microbial genomes by transferring genes coding for product-specific enzymes or entire metabolic pathways. The Escherichia coli bacteria and the Saccharomyces cerevisiae yeast were widely chosen as chassis for such genetic engineering and used as production workhorses.

Genetically engineering the metabolic pathways of microorganisms is only one lever in increasing production by fermentation. The time of clay pots maintained at constant temperatures used by ancient civilizations to ferment their foods is far gone. New analytical chemistry techniques such as high-performance liquid chromatography (HPLC) used to separate, identify, and quantify each component of a mixture increased the ability to modify both genetic components and growing conditions during fermentation to optimize the production of desired molecules and suppress byproducts. Implementing new gas sensors for real-time monitoring and new stirring and heating tools in precision fermentation vats allowed much finer grained control over growing and producing conditions.

All these advances opened the opportunity to produce more and better fuels, foods, and medicines using fermentation:

Fuels
The promise of producing fuel from arable lands instead of extracting it from the ground relies heavily on the ability to transform agricultural output into fuels at scale. The challenges have proven to be immense. Not only the price of the substrate (often glucose) is subject to external factors such as inclement weather and supply chain issues, but operational challenges from contaminations to endless purification steps to produce a relevant amount of products have been more problematic than anticipated. Of the many high-profile companies attempting to produce commodity products such as fuel in the 2010s, only a handful still exist. The difficulties in scaling up their operations have by in large either converted them into producing specialty products or made them disappear due to a lack of funding.

‍Foods
The need to rethink food and, particularly, meat production for ethical and environmental reasons pushed some entrepreneurs and scientists to look at producing proteins, fat, and other meat components in large vats, the way humanity has produced ethanol for millennia. While technically not within the broad definition of fermentation (as this work involves non-microbial cells), genetically engineering animal cells and growing them in vats using the same genetic tools and technologies as fermentation seemed like a solution for a hungry world. From better ingredients, to dairy products, to lab-grown meat, to cell-cultured human milk, to pet food, companies have demonstrated unbelievable ingenuity in scaling up production. Still, a damning report published in 2021 argued that the cost structures of these efforts would preclude the affordability of their products as food, plagued with the same challenging economics as fuels were before them. This hasn’t dampened the enthusiasm from industrial lobbying groups arguing for massive government subsidies to kick-start the capital investment needed. Nor has it apparently dissuaded the venture capitalists who funded cultivated meat companies with nearly $700 million in 2022.

Medicines
Recent advances in cell engineering have allowed microbial systems to slowly replace mammalian cell culture for the expression of therapeutic proteins. Glycosylation pathways have been engineered into microbial workhorses allowing for human N-linked and O-linked glycoprotein biosynthesis. These hosts have also gained popularity for antibody fragment production, which lack post-translational modification but still exhibit antigen-binding properties. Microbial organisms offer a simple, cost-effective system that offers an attractive alternative to animal cells.

Looking at a short history of fermentation, one can only marvel at how its development is intertwined not only with the broad biotechnology industry but with the enduring fight of humanity against hunger, disease, and climate change.  

– Steve Allen, Jonathan Friedlander, PhD & Geoffrey W. Smith



First Five
First Five is our curated list of articles, studies, and publications for the month. For our full list of interesting media in health, science, and technology, updated regularly, follow us on Twitter or Instagram.

1/ It’s all in your head
Can you heal with positive emotions? This might be a simplistic way to look at the problem but scientists are making progress in understanding the interplay between the nervous and the immune systems. An accessible review of their work is published this week in Nature.

2/ Hasta la vista, Baby!
Inspired by sea cucumbers (and most likely by the T-1000), engineers designed miniature robots that can shift between liquid and solid states. These tiny shapeshifters are magnetic and can conduct electricity. The researchers published how the robots can advance through an obstacle course and hypothesize on the different applications they could be useful for, from construction to drug delivery.

3/ Fewer calories, More years
In a large international randomized controlled trial, researchers showed that caloric restriction could slow the pace of aging in healthy adults. By measuring blood DNA methylation, scientists published that the intervention represented a 2 to 3% slowing in the pace of aging, which in other studies translates to a 10 to 15% reduction in mortality risk, an effect similar to a smoking cessation intervention.

4/ Understanding the rocks that made the (primal) soup
To recreate what might have been the conditions in which life first appeared on Earth, researchers used four-billion-year-old zircon chemistry to estimate the fluid chemistry and temperature for mineral formation. The models published in Science allow researchers to simulate what metals may have been transported to Earth's surface when life first emerged.

5/ Brain and gut
Mounting evidence links the microbiome to neurological diseases such as multiple sclerosis, or Alzheimer’s. A new study published in Nature Communications highlights how the gut microbiome is involved in multiple pathways in the pathogenesis of Parkinson's disease (PD). Through metagenomic analysis of the stool microbiomes of persons with PD and healthy control, scientists showed a vast imbalance in microbiome composition in people with PD.



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Public-Interest Technologies for Better Health

Digitalis Commons is a non-profit that partners with groups and individuals striving to address complex health problems by building public-interest technology solutions that are frontier-advancing, open-access, and scalable.

Establishing best practices are an essential part of building credibility and accountability for a movement. PLOS Biology published this month a concerted effort to agree on core open science practices. These may contribute to the development of policy, education, and interventions.

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