Ep. 2 Part 2: Milk w/o the Moo
By: Okezue Bell
Tags: Climate Change, Global Warming, Cows, Dairy, Cellular Agriculture

Ok, so BAUG the 🐮 is back from last time. Still alive, because we used cell culture and didn’t have to kill her for 🥩. Now, she’s being held hostage by these farmers holding her hostage in these horrible tie-stall barns! Her and her family are now being chained down, and are held in rows.

tie-stall farms

Unfortunately, this is a reality for millions of 🐄s. They’re not allowed to do their cow things because of their milk.

Now woah, woah, woah. I’m not saying that we should milk cows. From an evolutionary perspective, the point of bovines is to just reproduce and live. As such, you might think that milking cows is a good thing.

And it is. Dairy is great.

It’s not about us milking cows. It’s about how we’re milking cows.

The dairy market is valued at around $330 Billion, and every decade, dairy products, grow by 18% in their carbon footprint, while average cow herds scale up by 11% in the same timeframe. Annually, the milk yield increases by 15%, with total dairy greenhouse gas (GHG) emissions reaching a project 38% increase.

Then, if we look at milk, its one of the most inefficient technologies in the world. It’s only 3% efficient. However, its the fact that we’re not tangibly impacted by the fact that for 100 kg of food we give the cow, we get 3 kg back. So, it just happens that BAUG the cow can produce milk, but they’re not optimized to do so for commercialization. With this, we get a ton of GHGs.The world dairy sector releases an estimated ± 26% of total anthropogenic (GHG) greenhouse gases. Without proper sustainable development innovations, this number is expected to rise to 33% in the coming years.

The dairy production pipeline, showing the GHG contributions of carbon dioxide (CO_2), methane (CH4), and nitrous oxide (N2O) by dairy. A spike in the 21st century indicates a projected growth of dairy emissions.

In fact, if we look at the emission intensities of dairy vs other products, you can actually see that dairy generates a 35X larger proportion of greenhouse gas! The cheese ratio to GHG is 5.9 kg of CO2-Eq/kg, whereas peanut butter 0.17 kg of CO2-Eq/kg, meaning associated GHGs of dairy products are about 35 times greater than non dairy products. That’s crazy! Just look at how we milk BAUG!

cows milked

Cows are kept restrained from pastures using the zero-grazing method, effectively introducing them to an array of welfare issues. Cows become lame from laminitis, where their hooves swell, can suffer mastitis, an udder infection that causes 16.5% of all cow deaths, and become infertile, which happens to over 13% of cows in the US, stopping them from completing their evolutionary function of reproduction.

With zero-grazing (a.k.a. cow abuse 😒), a majority of cows are kept in tie-stall barns. The flooring damages their hooves and can cause extreme bone and muscle pains, increasing the rate of lameness, and they also have a high risk of infection. This is an unfortunate reality for over 40% of cows.

Fortunately, this is banned in the UN, but we also inject cows with a whole bunch of stuff. Growth hormone treatments are currently being used on livestock, which can heavily increase the associated risks of dairy product consumption, and can stimulate unnatural responses in the human body. For example, the recombinant bovine growth hormone or somatotropin (rBGH/rBST) is a growth hormone derived from the somatotrophin hormone found in the pituitary gland, which is a factor to promote grown and cell replication.

The rBST is engineered through editing the somatotrophin gene to create a hormone that promotes growth. rBST has been under further scrutiny, which has revealed concentrations of insulin-like growth factor 1 (IGF-1), a substance which, when in high enough concentrations, can inadvertently promote the development of tumors in the body. The overconsumption of milk specifically from bovines who were growth hormone treated can lead to higher levels of prostate, breast, and colorectal cancers. So our current milk treatments are detrimental to the cow, and potentially us.

The cows are experiencing a genocide.

On a better note, we have a really simple solution. With this:

Yep, that’s yeast.

Yeast. Remember bread 🍞? Well, guess what causes it to rise. Fermentation and yeast, which we’ll get to in a second.

Anyway, yeast is a type of fungus. Yes, yes… some people consider this mycoprotein (fungus based protein), but its really just acellular agriculture. HAHAHA 😈! Did that confuse you? We’re talking about cellular agriculture and I’m over here saying “acellular”. Well, acellular agriculture is a subtype of cellular agriculture.

The reason its called acellular is because we’re not producing cells as the final product. With things like meat and fish, we’re using cells to make cell products — like muscle tissue (myocytes/muscle cells). However, with things like dairy and leather, we’re using cells to make things like proteins (not cells), fabrics, and other products.

And this technology is already being implemented. Just ask Perfect Day, a super cool (and aesthetic) company leveraging acell ag technology to produce a variety of dairy products, from cheeses to butters to creams.

So let’s go over how this is do- But first, we’re going to have to talk about fermentation and respiration.

The -ation Cell Processes

So, there are two process (technically three) that we’ll go over: fermentation and respiration. These two are quite related actually, and inform the sustenance of the cell. Respiration is a process that’s essential to life, and is a relatively intricate (but can be explained simply). Same thing for cellular fermentation, which serves to power acellular agriculture. So, lets hurry up and explain this so we can get to the fun stuff!

The Process of Cellular Respiration

There are two types of respiration: aerobic and anaerobic. By their names, you can tell that one requires air (well, not air, but rather diatomic oxygen [O2]), and thus is called aerobic, while the other doesn’t require oxygen, so is called anaerobic. We’ll go over aerobic respiration first.

The process of cellular aerobic respiration is shown below:

As you can see, aerobic respiration starts with a process called glycolysis. Glycolysis is a anabolic process that breaks down the hexose (containing 6 carbons) glucose. First, glucose is turned into glucose-6-phosphate, which is sped up by the hexokinase enzyme.

Glycolysis — this won’t be very fun…

Maybe I should’ve explained this last time. Well, prepare for long names, but its not that confusing. Just long. 🧠 Let’s goooooooo! 👇🏾

Then, the energy currency of the cell, the ATP (adenosine triphosphate — don’t bother remembering that if you don’t plan on taking biology), is used for phosphorylation of glucose. Basically, in the name, you can tell that ATP is made from an adenosine molecule and three phosphates. What phosphorylation is is slapping a phosphate onto the glucose ring. From there, we lose one ATP.

Then we rearrange our G6P into F6P (fructose-6-phosphate) the reason this works is because F6P is an isomer of G6P, meaning it has the same molecular constituents (so the same formula), except with different arrangements. Because of this, if we rearrange the G6P, we can end up with a different sugar, fructose + 6P. Oh, and all of this is sped up by another enzyme called phosphoglucose isomerase (PI).

Then, with the help of the enzyme phosphofructokinase, we get fructose 1,6 bisphosphate from the original F6P. And another ATP slaps on a phosphate group for phosphorylation.

Then, there’s an enzyme adolase that helps to speed up the process that splits the bisphosphate into these really long-named molecules (whoever, named these…) called dehydroxyacetone phosphate (DHAP), and glyceraldahyde 3-phosphate (G3P or GAP). Don’t even bother remembering names (unless you’re taking a bio course, or its your profession 🙃). This is called cleaving by the way, which just means the cutting of a compound or bio-thing.

And then TIM over here 👉🏾 👦🏾 (real name: triosphosphate isomerase enzyme…) helps to turn G3P into DHAP and vice versa. Yes, these two are also isomers.

Then we get Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to slap on a Pi, or an inorganic phosphate, so we get 1,3-bisphosphoglycerate from G3P. Basically, a hydrogen is removed (dehydrogenation/ase), so we also end up with one of the H molecules, and important compound called NADH (ill explain why a little later. Hang tight!).

Then there’s Mrs. Phosphoglycerate Kinase. She loves slapping things with phosphates too!She transfers a phosphate group from 1,3-bisphosphoglycerate to ADP to form ATP and 3-phosphoglycerate.

BTW, ADP is adenosine disphosphate. It’s ATP missing 1 P. If it misses another, its adenosine monophosphate. Loses another? Its just adenosine.

Through phosphoglycerate mutase, the P from 3-phosphoglycerate is snatched, and 3 becomes 2. Therefore, 2-phosphoglycerate is formed.

Man, I’m thirsty…let me get some water🚰.

I found out that I was probably thirsty (ok this isn’t how it works but it’s a joke so calm down) because Mr. Enolase stole a whole water molecule 2-phosphoglycerate to make phosphoenolpyruvic acid (PEP) 🙄.

How rude of enolase. Because of him, I don’t want to talk about glycolysis anymore. Good thing we’re at the end. The last step is getting the pyruvate, two 3-carbon molecules by equally splitting one glucose (which recall, has 6 carbons). This is done thanks to pyruvate kinase 🤩, which snatches a P from PEP and gives it to the ADP, creating ATP and Pyruvate/pyruvic acid!

And BOOM! The whole process gives us 2 pyruvate and 4 ATP, but 2 were used, so we get a net total of 2 ATP. Anyway, I really want to highlight these processes in depth, so that you really understand why fermentation is so cool.

Let’s get to respiration, so then we can do fermentation, then the FuN sTuFf!

Respiration

Aerobic 👇🏾

So we already know that we start with glycolysis in the cytosol, the liquid part of the cytoplasm.

We then do pyruvate oxidation in the mitochondrial matrix, which is the inner part of the inner membrane of the mitochondria:

So, there are basically three steps of pyruvate oxidation. First, a carboxyl group (functional group with formula -COOH) is removed from the pyruvate. Then, a molecule of carbon dioxide (CO2) just floats away, and pyruvate is now a two carbon molecule called acetyl (group).

Then, we push some oxygen on that acetyl, oxidizing it, duh. Oh, and some electrons are left behind, which are swept away by the housekeeper NAD+, which then becomes NADH after interacting with the electrons.

Finally, the acetyl is smacked into coenzyme A. The acetyl and coenzyme are fated lovers and forms Acetyl CoA (Read: acetyl, coenzyme A). The marriage officiant in all of this is the enzyme complex pyruvate dehydrogenase complex.

Got to love some good old bio-Romeo and Juliet. Except w/o all the suicide, poison, Capulet, Montague, etc., etc.

Instead we have enzymes, NADH, and coenzymes. So, through the oxidation process, we end up with 2 NADHs and Acetyl CoA. Next up:

The Kreb’s Cycle

From there, we move on to the Kreb’s Cycle. This is also known as the citric acid cycle or the tricarboxylic acid cycle. If I were you, I’d stick with “Kreb’s”.

It consists of a series of steps that can be easily described through bullets!

Acetyl CoA (2 C) smacks carbons with (4 C) oxaloacetate and creates citrate (not to be confused with citrus/citric acid. That’s what’s being created). You know, the stuff in your orange and/or pomegranate juice. We get isocitrate by moving around the molecular configuration of citrate (its an isomer of citrate). The isocitrate is converted to α-ketoglutarate! Thanks isocitrate dehydrogenase! α-ketoglutarate is oxidized. Now, it has 4 carbons, and of course it runs to coenzyme A. From there, we get the acetyl CoA ripoff: succinyl CoA (very important though lol). We also get some NADH and CO2! Succinyl CoA is whipped into shape to create succinate, which also has 4 carbons. A molecule called guanosine triphosphate (GTP) is also produced as a byproduct. Then from succinate, we get fumerate, its (still) 4 carbon isomer. We also get another derivative molecule from that called FADH2. Fumerate → malate (ANOTHER 4 CARBON isomer?!?!?!?!) Malate → oxaloacetate + more NADH

… — that was my reaction when I first learned ALL of the steps in the Kreb’s Cycle. Not very exciting, huh?

Well, at least we get 2 CO2, 3 NADH, 1 FADH2, 3 H+, and 1 GTP from it. That’s something Bio, Honors Bio, and AP Bio tend not to tell you… its not all about the FAD-NAD-CO2, is it? btw bio classes are great… no shade. The last stage?

Oxidative Phosphorylation

This is basically oxygen-based-slap-a-phosphate-group-on, and happens inside of the mitochondria’s inner membrane.

The first step to doing this is a redox reaction. This is reduction-oxidation for short. Basically, its just a bunch of electron transfers. There are specific membrane proteins in the mitochondria moving the electron around.

The protein losing the electron is considered oxidized, while the protein receiving the electron is reduced. Through these processes, the electron transport chain (ETC) functions. That, combined with chemiosmosis (chemical based osmotic diffusion… get to it in a sec 👍🏾) makes up oxidative phosphorylation.

And yes, I intentionally dropped some buzzwords hoping that you would be interested in what they mean and how they work.

In the ETC, those molecules I mentioned before, NADH and FADH2, are important because they are electron donors. They deliver their electrons and become reduced, meaning that they turn back into NAD and FAD respectively.

The delivered electrons release energy as they move energy levels, and this energy can be used to push out some protons (H+/hydrogen ions), which shoves them out of the matrix and into something called the intermembrane space (recall: its the in between area). The pump creates a gradient, which is basically a gradual change in energy. The gradient is called electrochemical for obvious reasons (just look at it; involves electrons and chemical properties…).

The chemiosmosis part allows for these proton movements across a hydrophobic wall anyway:

I’m thirsty again (again, a joke, not actually how it works) and want some 💧.

Fortunately, we’re at the point where oxygen, which exists diatomically in nature (x2 bonded together) as O2, but its split in halves, both of which bond with H+ to create some H2O 💦.

The proton motive force means that hydrogen protons are moving down the gradient, and they pass through the final enzyme, ATP synthase, and through the last electron acceptor, oxygen, the energy is used to create ATP!!

And there you have it. Aerobic cellular respiration.

Now let’s talk about what you do without oxygen, and what you remove.

Anaerobic Respiration

The process of anaerobic respiration isn’t very difficult to explain, considering its basically a truncated version of aerobic respiration!

It’s typically completed by bacteria and archea.

So, glycolysis doesn’t require oxygen, so that can happen, and the Kreb’s cycle kind of doesn’t require oxygen per se either, but without oxygen, it stops, because NAD and FAD run out. So I guess it does require oxygen, but scientist like to make things confusing sometimes so they say that it doesn’t 🤦🏾‍♂️.

Though oxygen sits at the end of the electron transport chain as the final electron acceptor to allow for oxidative phosphorylation, organisms like sulfur reducing bacteria don’t have to use oxygen as their final electron acceptor for the ETC. SO42− (sulfate), for example, can be a substitute, along with nitrogen, sulfur, and others. When a different electron acceptor is used, there is a different molecule released. With oxygen, we got CO2, but with sulfate, we get H2S, or hydrogen sulfide.

However, there is another important anerobic pathway that dies directly into how we synthesize dairy without the cow:

Fermentation

There are two main types of fermentation: lactic acid and alocholic/ethanol.

Fermentation begins with glycolysis, except when we make pyruvate this time, there’s no ETC, or Kreb’s Cycle, or oxidation, or any of that jazz. Instead it goes through a different pathway system which ultimately creates NAD+ from the NADH in glycolysis, and this regeneration allows for glycolysis to reocurr.

So, let’s start with lactic acid fermentation.

Ever had that burning sensation in your muscles while doing any exercise, like running? Well, thanks to lactic acid fermentation, you get this sensation because lactic acid builds up in your muscles. As you breathe heavier, you intake less oxygen, meaning that your body has to rely on fermentation to continue glycolysis!

Side note: don’t confuse this for tiredness or muscle soreness. Lactic acid buildup that effects you typically occurs at the point at which you are exercising really hard and then your myocytes don’t get enough oxygen.

So, this is what lactic acid fermentation (LAF) looks like:

Basically, glycolysis occurs as it normally would, where 2 NAD+ are put in to give 2 NADH and also 2 pyruvates from the broken down glucose. However, instead of moving the the ETC like we saw before, the pyruvates are instead converted to 2 lactic acid molecules, which are then ionized to form lactate! In the meantime, 2 NADH is oxidized into 2 NAD+, which allows for glycolysis to occur again.

Now, what about alcholic fermentation 🍾🍷(***the other*** AF).

It looks like this:

Same as lactic acid, except were producing the ethyl alchol instead! So we go through glycolysis, then we use the 2 pyruvate to create 2 acetaldehyde (a type of compound), which releases carbon dioxides. Then, the 2 acetaldehydes are converted through NADH to two ethanol alcoholic compounds, which then oxidizes the NADH and turns it to NAD+, which is used to power glycolysis again. Now, let’s apply this to dairy.

The yeast undergoes the alcohol fermentation process to actually create the two proteins that basically define milk. Let’s take a look.

Making Milk w/o BAUG the Cow

BAUG is 2320 lbs. We don’t need her for milk anymore. We’ll continue to milk her to keep her healthy, but in terms of the dairy business, we don’t need to drain her dry.

But, we do need her proteins and genes, which is nothing compared to over-milking her for a year to only get 3% of the resources we put in as milk/dairy…

So milk. It’s not very complex:

And that’s it.

The main proteins inside of milk are called casein and whey. There’s about an 80–20 percent ratio of them inside of milk. Casein and whey are considered very high quality proteins, as they’re composed of extremely essential amino acids in high quantities.

Then there’s lactate or lactic acid (same thing), one of the primary nutrients inside of milk. And then your classic H2O.

So if milk has such simple products, we should be able to recreate it in a lab, right?

Right.

And that’s where cellular agriculture comes in.

Cultured Milk

Let’s talk about how cultured milk is made. Instead of taking BAUG and separating her and her child, and then hooking her up to a bunch of machines that will milk her, we can just be smart about it and make what we need from milk’s base components.

Basically, without this technique, BAUG would have her children taken away, be milked for a year, only convert 3% of fed nutrients to milk for a year, become pregnant, and then have her baby calf snatched again… and then the cycle repeats 😢.

In short, this is what we’re doing.

This is what we should be doing.

However, cellular agriculture can once again change everything: much less environmental impact, and more cow welfare. The process works like this:

This all starts with GM (genetically modified) yeast.

Making GM Yeast

So what we do is give the yeast the proper genes so that it can begin producing caesin and whey just like the cow. These proteins inform milks taste, color, and properties the most, so they have to be present when reproducing milk synthetically. Basically, after identifying the gene in the cow that makes the proteins, we (harmlessly) get it and introduce it to the yeast’s DNA. Then we get the plasmids (recall: circular bundles of DNA) to continuously replicate. This process is sometimes called cloning. It functions essentially like this:

plasmid cloning

Because the plasmid never stops replicating, it is the site of the insertion of, for example, the kappa-casein gene, which is one of the most vital genes for protein production in milk-yielding bovines. By the way, the plasmid is called recombinant after it’s gene edited. The plasmids are essentially new gene DNA factories.

Basically, we cleave the gene that we want to replace, and then we insert the new one! Then the plasmid is put back in the yeast so that the plasmids can tell the chromosomes what’s going on, so that the chromosomes (which are the main sites of DNA) assume that gene as well. This is called transformation.

Then when the yeast splits, it produces casein and whey! The process is shown below:

Ok, so we now have some great lab grown yeast. Perfect! However, if everybody drinks about 199 lbs. of milk every year, then we’re going to need wayyy more proteins.

This is were the process of fermentation comes in.

Remember bioreactors? Well, they’re still useful.

But remember, there’s still a lot of scaling that needs to be done with them.

Anyway, the protein-producing yeast is put into these tanks where they continue to duplidivide (duplicate + divide). This allows for the mass production of casein and whey.

The bioreactors provide a large and stable surface environment for fermentation to occur, so that the yeast is grown to produce casein and whey. For this reason, bioreactors are sometimes considered fermentation vessels, as they serve a similar purpose to a beer growing keg.

Within the vessels, the yeast is provided with everything it needs to keep producing casein and whey, and the plasmids are informing everything on the cellular level of the genetic information change.

From there, purification occurs, where casein and whey are efficiently pulled away from the yeast so that the two proteins can go on to become anything milk-based: