# Why is fermentation of cellulose to produce biofuel and nutrients so difficult?

The formula for glucose is $\ce{C6H12O6}$ and that of cellulose is very similar $\ce{C6H10O5}$. Glucose can be readily fermented by yeast and other micro-organisms to produce carbon dioxide and ethanol. Whereas cellulose is not ferment-able by yeast and only very specific anaerobic Archae bacteria can digest it, but the end product being methane, water, sugars (primarily lactose), and volatile fatty acids (acetate, proprionate, butyrate); not ethanol. These bacteria reside in the guts of termites that allow them to digest wood, and in the intestines of cattle and other ruminants. The termites and ruminants use the sugars and fatty acids as food.

The enzymes used by the host anaerobic bacteria in the gut of termites are of particular interest because:

• termites produce very little solid waste as feces
• termites can digest wood, leaves, and broader variety of plant fodder than ruminants, which mainly feed on grasses

We are not really interested in the termites, nor the anaerobic bacteria, but rather specifically the enzymes and chemical processes used by the bacteria that break down the cellulose into the lactose and fatty acids. Can this be accomplished synthetically in an anaerobic lab setting outside of said bacteria?

This article describes a company that tried to use cellulose raw material to produce ethanol on a commercial level, but recently went bankrupt. This company apparently was not getting ethanol directly from fermentation of cellulose but rather the result of a multiple step process of generating syngas from the cellulose, then creating alcohol from the syngas; a much less efficient process than direct fermentation of cellulose into alcohol.

Obviously, such a process if achievable would relieve the need to use food crops to generate ethanol and put to use otherwise inedible cellulose plant waste. What is the main challenge here. Cellulose is just one water molecule different from glucose.

• Cellulose was designed so as not to be easily digested. No one likes to be someone else's food. – Ivan Neretin Oct 10 '17 at 4:51
• The gist is that starches are essentially linearly polymerized glucose. Bacteria can easily unzip starches. However cellulose has crosslinks and thus can be unzip as easily. – MaxW Oct 10 '17 at 5:55
• @IvanNeretin So... creationism? – vapid Oct 10 '17 at 10:40
• @vapid I never said the design was intelligent. On the contrary, it was pretty dumb (otherwise it could have come up with something like kevlar instead). But it works. – Ivan Neretin Oct 10 '17 at 11:04
• @IvanNeretin I bet that the dumb Nature would have came up even with a 'kevlarase' if necessary. – vapid Oct 10 '17 at 11:43

Your question is a little bit all over the place, but I believe I can answer it anyway. First, though, allow me to point out that your sum formula for cellulose is wrong. While glucose is indeed a single $\ce{C6}$ compound with the exhaustive sum formula of $\ce{C6H12O6}$, cellulose is, in fact, a poorly defined polymer consisting of multiple $\ce{C6H10O5}$ subunits, linked together both linearly to form long chains but also cross-linked to form extensive networks. Therefore, the better sum formula would be $\ce{(C6H10O5)_nH2O}$ or $\ce{C_{6n}H_{10n+2}O_{5n+1}}$ for very large values of $n$.

There are two reasons why glucose, its dimer maltose and its polymers amylose, amylopectin and glycogen are much more easily digested by all organisms than cellbiose (the glucose dimer that can be found in cellulose) and cellulose itself.

The first one is the type of linkage: the first set of compounds all contain α$\ce{{1}\bond{->}4}$ connections while cellbiose and cellulose contain β$\ce{{1}\bond{->}4}$ connections. This seemingly minor difference means that while the starch subunits form spirals, the cellulose subunits actually form fibres and all the enzymes designed to break up spiral subunits cannot work with fibres.

The second is the number and type of cross-linkages. Each organism has a set of enzymes that can break up the cross-links present in amylopectin or glycogen (or both) but these again are different in shape from those in cellulose owing to the subunits’ β configuration in the latter.

Enzymes to break down amylose, amylopectin and glycogen are pretty much present in every multicellular organism since these compounds are used to store energy in an available form — which would not make sense if the organism could not break the storage form back down to monomers. However, cellulose was not ‘intended’ to be energy storage and thus there was no evolutionary pressure to evolve enzymes that could digest it. Therefore, only very few species that found and populated this ecologic niche are able to digest cellulose and even less are able to digest the additional compounds present in wood that makes it hard.

Now on to the process design part of your question. In general, if we have the enzymes we should be able to degrade wood to useable monomers much like whichever organisms do that in whichever environment they live. Not only the symbionts of termites are of interest but also wood-digesting fungi, naturally. However, the key difficulty here is acquiring the enzymes required in sufficient amounts to make the process economic. I am only aware of very few enzymes that can be fermented and isolated in sufficient quantities and purity to make for a possible economic use; among them a generic lipase. That is one significant hurdle process chemists must overcome.

However, there’s another bit. It may work if we have access to the enzyme but it doesn’t have to. A number of times the most significant issue for chemical engineers is the scale-up problem: reactions work fine on a small scale but once you try to scale them up to higher quantities for more throughput they break down in one way or another. I am sure without knowing the exact titles that there are shelves of books concerning the scale-up problem. To the best of my knowledge (caveat: I am not an industrial chemist or chemical engineer), it is often down to trial and error which processes work well and which don’t; and what works well for one type of product does not work at all for others.

Needless to say that one has to have access to a significant amount of funds to bridge the research gap until one actually is able to make money so it doesn’t surprise me (although I am unhappy) that the company you mention went bankrupt.

The ultimate reason is that cellulose is designed to be hard to digest

The specific chemical reason is well covered in Jan's answer, but there is an explanation that is simpler and more fundamental: cellulose is designed that way. Nature has created some organisms that need a structural component that isn't easy for other organisms to break down (for the avoidance of doubt evolution sometimes produces outcomes we call designed we don't need an external designer).

Trees want to last for a long time and, if other organisms could digest their structural material easily, they wouldn't achieve that goal. Cellulose is one of the key structural compounds enabling tree trunks to be strong and long-lasting. If they were built from a different sugar polymer like starch, they would be easy to digest (starch is designed to be a storage medium for sugars and is relatively easy to convert back to digestible forms which is why a lot of our foods are mostly made from it).

So, ultimately, the indigestibility of cellulose is a product of an evolutionary history where some organisms want to be big, strong and long-lasting. Very few other creatures have evolved good ways to beat this property (the symbiotic bugs in termite stomachs are a rare example).

The main challenge is the macroscopic structure of cellulose. It's a solid, enzymes obviously can only start working on the surface, and hydrolases will work only on a few random loose chains.

This is too slow, even if you have a lot of time and are not trying to make any money. So you need to break apart your cellulose first using a process that's cheap and results in a product clean enough for the enzymes (you can use acids, but not too much or you'll prevent the enzymes from working). This is not too easy.

After you break the crystalline structure you need to use an enzyme mix, as you need still need some more "starting points". You need enzymes to make some breaks in the middle of the chain, some enzymes too loosen the chain from the bulk, etc. before you can use an enzyme that digests cellulose from the end (like happens easily with starch). The hydrolases are fast enough, but you need quite a few more activities.

Then all of this needs to fit together, you need a source that's really really cheap, a process that breaks it down enough for the enzymes to do their work, and then this has to result in a mix of glucose and other compounds that are good food sources for the fermentation step, and this has to contain no competing micro-organisms or other inhibitory stuff. And remember, all this time you're competing against dirt-cheap fossil fuels.

Creating some ethanol is trivially easy at the moment, you can order some enzymes, boil your cellulose in acid, wash it, add the enzymes, add some yeast and distill off your ethanol. But doing this in an economically feasible way is not that easy.

One company failing is also not an indication that it doesn't work / isn't feasible. Dupont for example is producing a lot of ethanol using an (partially) enzymatic process. You can browse around and get an idea of the challenges, bottlenecks and the scale of the operation. Other companies are also doing a lot of stuff, for example POET-DSM and Abengoa.

There are quite a number of cellulases in nature (enzymes to split cellulose into shorter oligosugars). See e.g. https://en.wikipedia.org/wiki/Cellulase

They are needed by any plant that grows, to be able to do some restructuring. They however only work on amorphous chains, and only one type (endocellulases) is actually able to cut into the middle of a long chain or network segment. The rest just bites pieces off (the resulting) dangling ends.

Crystalline segments (they're what makes trees so hard and elastic) are virtually immune to enzymes. They only degrade after previous e.g. oxidative or UV damage. Wood under water does not decay on human timescales, unless you go to higher temperatures and/or acidic conditions.

Your termites et al. have a lot of tiny, sharp teeth (figuratively speaking) and mechanically split the wood into very small particles, the huge surface of which allows the enzymes to get to work again.