L20. Pathways to Products: Fermentation
Introduction
In the last two lessons, we learned about agriculture and what it takes to produce the crops that will serve as biomass feedstocks. But now what happens? How do we convert the biomass we grew into chemicals and fuels?
A variety of processes have been developed, and we can organize them into three categories:
- Biochemical processing - uses enzymes (biological catalysts Links to an external site.) and microorganisms (e.g. yeast, bacteria) to convert carbohydrate-rich biomass into desired products
- Thermochemical processing - uses heat and catalysts to convert lignocellulosic biomass (the cellulose/hemicellulose/lignin part of the plant) into products
- Processing of oleaginous (oil-rich) biomass
This lesson will cover only biochemical processing; thermochemical processing and processing oleaginous biomass will be covered in the next lesson. The biochemical process we are going to focus on here is fermentation. You can think of fermentation as sort of a magical black box - we put biomass in, and out comes nearly any bioproduct we can dream of. Some you are very familiar with - wine, beer, cheese, sauerkraut, yogurt, while others such as vitamins, enzymes, acids, and pharmaceuticals you may not have heard about.
Getting sugar for fermentation
Defined simply, fermentation is the chemical breakdown of a substance by bacteria, yeasts, or other microorganisms. Fermentation most commonly takes place in the absence of oxygen, otherwise known as anaerobic conditions. The “substance” that gets broken down is a six-carbon sugar (like our fundamental friend from Lesson 9 - glucose) or a disaccharide (e.g. sucrose). So if we want to use fermentation to produce a wide variety of chemicals, we first need to convert the biomass we’ve grown into sugar.
From sugar crops
The type of plant we get our sugar from depends on economics and climate, which means it varies based on where you are in the world. Sugar beets are popular in Europe, while sugarcane is important in Brazil. Both of these plants are called sugar crops and it’s exceptionally easy to get sucrose out of them. Let’s use sugarcane as our example. 
Traditionally, the leaves and tops of the sugarcane plant are burned or left in the field, while the stalk is transported to the sugar mill. The cane is then milled to separate the sugar from the fibrous part of the plant. The cane is chopped and shredded with revolving blades, and washed with hot water to produce a sugar-rich juice and a leftover fibrous residue, known as bagasse. Bagasse can be burned as boiler fuel, and is the primary source of electricity used by Brazilian ethanol plants. The sugar-rich juice is then filtered multiple times and evaporated until a mixture of sugar and molasses remains. Mission accomplished! The sugar goes on to be fermented and distilled to produce ethanol, but we will cover these steps in more detail later. Note that the sugar-rich juice can also go on to be processed into sugar for the food industry. The flowchart below is a conceptualization of the steps we just described.
Figure 1. Flow chart for ethanol, energy, and sugar production from sugarcane (Di Nicola 2011 Links to an external site.)
From starch
In the United States, our main source of sugar (specifically glucose) for the production of bioproducts and biofuels is corn. In Lesson 18, you watched a video that showed all of the various products that corn can be turned into. Now it’s time to dig into the specifics of how these conversion processes actually work.
Remember from Lesson 9 that both starch and cellulose are long chains of glucose molecules. 
Which means we can get our glucose for fermentation from either the starchy parts (the kernels) or the cellulosic parts (the stalks and leaves - which we’ll call stover from here on out) of the corn plant. The process for extracting sugar from the kernels is different than that from the stover, so we will discuss them separately. First, let’s talk about the kernels.
The vast majority (upwards of 99%) of corn-based bioproducts and biofuels come from the starch stored in the corn kernels. This is because the bonds within a starch molecule are easily penetrated by water, making it very easy to break starch down into usable glucose monomers. Since glucose from starch is so readily accessible, it is the efficient, economical choice for a bioproduct/biofuel feedstock.
The first step on a corn kernel's journey to becoming a bioproduct or biofuel is milling, and there are two types: dry and wet. Of all the ethanol fuel produced in the US, 80% comes from dry milling, so we will focus on that here. Dry milling is essentially a simple grinding procedure where kernels are ground in a roller mill into the consistency of cornmeal (here’s a 57-second video
Links to an external site. of grinding corn if you’re interested). Now that the corn 
has been broken down to expose the starch, it is mixed with water to form a corn slurry. The slurry then goes through cooking and liquefaction. If you’ve ever cooked with cornstarch, you know it’s used to thicken things like pie filling and gravy. The same thing happens here - as the slurry cooks it becomes very thick and viscous. In liquefaction, an enzyme called amylase is added in, which breaks down the starch molecules (polysaccharides) into smaller maltose pieces (disaccharides). The next step is to break the maltose down into even smaller pieces - our glucose monomers - using another enzyme in a process called saccharification (a fancy way of saying sugar-making). And just like that, we’ve got the glucose we need for fermentation! All it took was two enzymes and a little heat - easy peasy.
Figure 2. Dry milling of corn (Brown & Brown 2014)
The capital investment for dry milling is less than that for wet milling, because the wet milling process is substantially more complicated. We’ve included a diagram of that process below if you are interested, but you will not be quizzed on it. The important thing to know is that wet milling separates the corn into its individual components: starch, corn oil, gluten, and hulls. This is advantageous because it gives a company access to higher value markets and provides flexibility in what products they can create (i.e. the starch can go to food or fuel production).
Figure 3. Wet milling of corn (Brown & Brown 2014)
From cellulose
As we’ve learned, it’s relatively easy to get sugar from starch. That is not the case with lignocellulosic biomass, like corn stover. Again, think back to Lesson 9. The cell walls of plants are made of cellulose embedded in a lignin-hemicellulose matrix. The whole purpose of these walls is to provide the plant with strength and structure, so it makes sense that they would be very difficult to break down. A variety of processes have been developed to separate lignocellulosic biomass into its three parts: lignin, hemicellulose, and cellulose. In our example of corn stover, 38% of the biomass is cellulose, 26% is hemicellulose, and 19% is lignin (source
Links to an external site.). Once separated, cellulose can be further broken down into glucose which can be used in fermentation. Hemicellulose, on the other hand, can only be broken down into 5-carbon sugars which are difficult to ferment. Lignin is a super complex polymer (not a carbohydrate like the others!) that we really don’t know what to do with, so we usually just burn it as boiler fuel.
Pretreatment is the first step in the conversion of lignocellulose to sugars, and is one of the most expensive. An important goal of all pretreatment methods is to reduce the size of the biomass material and increase the surface area. Primary size reduction is achieved mechanically by sending the cover stover is through a hammer mill (similar to corn grains). Secondary size reduction can happen in a variety of ways including biological, chemical, and physical approaches. Some examples include: using microorganisms to decompose lignin (thereby releasing hemicellulose and cellulose), using alkali metal hydroxides to dissolve lignin and hemicellulose, and using steam or liquid ammonia to explode the biomass into separate fibers. The pretreatment step that is chosen depends on which subsequent hydrolysis methods is to be used (described below).
The next step in the process is hydrolysis - the chemical breakdown of a compound due to reaction with water (both liquefaction and saccharification were examples of hydrolysis). In the case of corn stover, we are trying to break cellulose down into glucose sugars. Hydrolysis can be achieved with chemically with acids or with enzymes. With acid hydrolysis, very little pretreatment is required besides grinding the biomass into tiny 1 mm pieces. Enzymatic hydrolysis, in contrast, requires extensive pretreatment to separate the lignin, hemicellulose, and cellulose components. The main problem with acid hydrolysis is that it requires either a large volume of acid, about equal to the weight of the sugar produced (which is costly), or really high temperatures (which reduces the yield of sugar). The issues with enzymatic hydrolysis is that it requires a whole cocktail of enzymes (instead of just two like with starch) and can be a slow process (taking up to 7 days to digest lignocellulose).
You can see how complicated it is to convert cellulose to sugar, making it a less efficient, more costly source of glucose than starch. However, there is a lot of excitement about, and research behind, using corn stover to produce bioproducts and biofuel, and here’s why. First, we have A LOT of it. While corn stover is typically tilled back into the soil after harvest to replenish soil nutrients, up to 70% of it could be removed from the field while still preserving soil health. Therefore, it is an extraordinarily low cost feedstock compared to corn kernels. If we could figure out how to lower the costs of pretreatment and hydrolysis, we’d have a very cheap source of sugar on our hands. Second, we’ve learned about the environmental benefits of growing perennial crops like switchgrass and miscanthus as opposed to annual crops. If we made more biofuels or bioproducts from lignocellulosic feedstock, that would make it profitable for farmers to grow something other than corn. But for now, less than 1% of our ethanol fuel and other bioproducts comes from sugar derived from cellulose, it is almost all from the corn grain.
Now that we’ve extracted our simple sugars from either sugar crops, starch crops, or lignocellulosic feedstock, we can finally get to the step we’ve all been waiting for - fermentation!
Fantastic Fermentation
Take a break, stretch your legs, because this is where it really gets exciting. Using fermentation, we can take our sugars and turn them into a wide variety of bioproducts.
Fermentation can be carried out by a number of microorganisms such as yeast (like S. cerevisiae) and bacteria (like E. coli). These “host organisms” are chosen based on how efficiently they can make the product we want and how well they can handle the conditions of the production plant. For example, if a product can only be made at 50°C, but yeast is happiest at 30°C, we would want to pick a different host that can handle higher heat. This way we won’t degrade the product, or have to spend the money to cool it down.
Let’s start with ethanol fermentation as an example, because it’s the most straightforward.
Ethanol
As with all cells, yeast need energy to survive, and they produce it through a series of chemical reactions (collectively known as respiration). In anaerobic conditions, yeast create energy by converting glucose into carbon dioxide and ethanol in a reaction we call fermentation. Ethanol, then, is just a respiration byproduct that we capitalize on. Since yeast produce it naturally, all we need to do to generate ethanol is to provide the yeast with the proper conditions to grow. In a ethanol fermentation, we give the yeast the saccharified corn mash to serve as their source of glucose, as well as nutrients, oxygen, and the proper temperature and pH. As they grow and reproduce, they simultaneously produce the ethanol we want. When the ethanol concentration reaches the right level, we stop the fermentation and separate the yeast cells from the ethanol and other water-soluble products.
All products made from fermentation go through separation and purification phases to get them ready for commercial use. With ethanol, this phase is called distillation. Distillation is an energy intensive process where the solution is heated to different temperatures to evaporates the various chemicals and separate out the ethanol. The first distillation yields 55% ethanol and stillage bottoms (which are sold as animal feed because they are high in protein), and the second distillation produces 95-96% ethanol (190-192 proof). Essentially water-free (199+ proof) ethanol can be achieved with further distillation. Fun fact: this is the same process that's used to make vodka. Before it can be sold at the gas pump, the ethanol must be blended with gasoline. Most cars on the road today in the U.S. can run on blends of up to 10% ethanol. The graphic below shows the entire process from corn milling to stillage bottoms (distillers grains) and ethanol fuel.
Genetic modification
While it’s true that yeast naturally produce ethanol, the yeast used in modern ethanol production have been genetically modified to produce higher yields of ethanol, allowing us to get more bang for our buck. In fact, nearly every microorganism used to produce biochemicals via fermentation today has been genetically modified. Not only can we use genetic modification to get our host organism to produce our desired product more efficiently, we can also use it to get the microorganisms to produce chemicals they wouldn’t naturally make.
This is where the magic of science comes into play - using genetic modification, we can get our host organism to produce any bioproduct we want. Once we’ve found the gene that codes for the chemical we want, all we have to do snip it out and insert it into our host organism (e.g. yeast). As the yeast goes about its normal life feasting on glucose and reproducing in the fermenter, it simultaneously produces our desired chemical because it is now part of its DNA. This product can then be separated out and sold commercially. The first chemical we ever produced this way was insulin - and now millions of diabetics worldwide use synthetic insulin to regulate their blood sugar levels. Please look at this illustration from the NIH Links to an external site. and watch the video below to better understand how this technology works. There will be quiz questions on these resources.
So really, all we are trying to do with fermentation is make the genetically modified microorganism happy. If they are happy and well-fed, they will multiply, automatically producing the biochemical that's encoded in their DNA.
Other products
Because of genetic modification, the possibilities for producing biochemicals using fermentation are endless. Ethanol fuel? Fermentation. Pharmaceuticals? Fermentation. Enzymes in your laundry detergent? Fermentation. Flavor enhancers? Fermentation. Preservatives? Fermentation. Bioplastics? Fermentation. Get this - we even use fermentation to make the enzymes that break cellulose down into glucose used for fermentation.
Many chemicals can be made from biomass via fermentation, but very few are produced in large quantities, which we refer to as commodity chemicals (more than 50,000 tons produced globally per year). Besides ethanol, only a few other fermentation products can be considered commodity chemicals: monosodium glutamate, citirc acid, lysine, and gluconic acid. The uses of these chemicals and a select few others are summarized in the table below.
Table 1. Biochemicals produced via fermentation & their uses.
|
Bioproduct |
Uses |
|---|---|
|
Monosodium glutamate (MSG) |
A flavor enhancer commonly added to Chinese food, canned vegetables, soups and processed meats. |
|
Citric acid |
Flavoring and preservative in food and beverages, especially soft drinks and candies. Also cleaning products and pharmaceuticals. |
|
Lysine |
Animal feed additive, some medicines |
|
Gluconic acid |
Food additive (sour flavor), cleaning products, also used in construction, textile and pharmaceutical industries. |
|
Isopropyl alcohol |
Rubbing alcohol, hand sanitizer, eyeglasses cleaner, fuel additive |
|
Linoleic acid |
Oil paints & varnishes |
|
Sorbitol |
Sugar substitute, laxatives, cosmetics |
|
Succinic acid |
Dietary supplement, food additive, pharmaceuticals, detergents, bioplastics |
Conclusion
Fermentation is one pathway to get useful products out of biomass. In the fermentation pathway, biomass is converted from starch or lignocellulose to a microbial food source (sugar). Microorganisms use the sugar to grow, and as they are grow and multiply they produce the desired product.
That’s a wrap on the fantastic world of fermentation! In the next lesson, we will learn about other pathways we use to convert biomass into biofuels and bioproducts.