00:00:00 What are these isomers that we're talking about?

00:00:30 What are these isomers that we're talking about?

00:01:00 And that's the only difference. It's just one stereochemical difference.

00:01:05 Now, that difference changes the optical properties of the carbon atom to which that OH group is attached,

00:01:14 and makes the molecule shift the plane of polarized light to the left instead of to the right.

00:01:22 That's it.

00:01:24 And the same is true of all optical compounds, based on the mirror image nature of the two isomers that make up the racemic mixture.

00:01:36 How can that be? How can that not stop?

00:01:43 We were talking about just the small difference.

00:01:46 Yes.

00:01:48 Does this manifest itself as sort of left- and right-handedness with the optical shifts, I guess?

00:01:56 Right.

00:01:57 From little molecules to big ones?

00:01:59 Yeah, that's the basic difference. I mean, it's a question of mirror image, basically.

00:02:03 If you take the one form and look at it in a mirror, you get the opposite form. That's the L and the D.

00:02:09 And it's true of small molecules, like, say, glucose, but also of, say, amino acids, which are relatively small.

00:02:18 But you can string them together in long chains or in complex structures,

00:02:24 and each little unit maintains its specific optical property.

00:02:31 And the total optical property of a larger molecule reflects the sum of all these others.

00:02:40 So in nature, of course, we're stuck pretty much with one isomer.

00:02:47 The world has been so evolved that living things in general are composed of one of the two possible mirror images of the basic compounds.

00:03:01 We talked about the optical. What are the natural implications of this?

00:03:07 Why is this important?

00:03:13 I suppose the most important word would be specific. I mean, the whole problem in biology is specificity.

00:03:20 And since everything that goes on in living cells is carried out by enzymes, it's important that the enzymes see only one form.

00:03:32 As a matter of fact, if an enzyme works on one form, say, the correct L form of an amino acid, it will be inhibited by the D form.

00:03:41 The D will be able to occupy that same site on the protein enzyme

00:03:47 and will prevent the interaction of the correct form with the active site.

00:03:53 So the whole system of life is integrated into this selection of one form of each basic molecule.

00:04:02 I've heard of hand-in-glove analogies, block-and-key analogies. Are those analogies?

00:04:08 That's basically what I'm saying, yes.

00:04:11 The lock-and-key situation is designed so that only one form will fit the other form, and you have specificity.

00:04:22 And that's precisely what happens in all of these biological systems.

00:04:27 I mean, these days, one of the hottest areas in biomedicine is the question of receptors on cells.

00:04:35 Receptors will recognize and bind circulating materials and depend entirely on this property of specificity of shape and optical activity.

00:04:50 The optical activity goes along with shape. I mean, it's left or right, that sort of thing.

00:04:55 So the natural world just doesn't work right or wrong?

00:04:59 No. I mean, things would stop dead if all the glucose in the world suddenly became the wrong isomer, etc.

00:05:09 I guess the question is, how do we know that that's true, and how do we find out about it?

00:05:18 Well, it was observed—actually, Pasteur did some of the most critical experiments with tartaric acid,

00:05:26 which is, I think, a component of wines, and it can form three forms.

00:05:31 It can form an L-form, a D-form, and a meso-form, which is a mixture of the two.

00:05:39 And he was able to show that he could crystallize one of the isomers out at a time, separately,

00:05:48 and that they not only crystallized out as though they were different substances,

00:05:54 but also, in his work and some later work that was done, would rotate the plane of polarized light either to the left or to the right.

00:06:03 And he found that the naturally occurring material was Levo.

00:06:08 That was quite early in the business.

00:06:11 And since then, it's been shown for all sorts of molecules.

00:06:15 In proteins, of course, which is my own particular interest, it's perhaps most dramatic

00:06:20 because these are very, very large molecules containing hundreds of individual amino acid residues, each one of which is L.

00:06:29 And the whole business of protein metabolism and protein synthesis

00:06:34 and most of the DNA recombinant methodology depends on the fact that these are always the same.

00:06:42 They can be depended upon to be L.

00:06:45 Now, how this started in the first place is anybody's guess.

00:06:49 It's been assumed that some naturally occurring minerals, for example,

00:06:57 might have been involved in binding one form and not the other form

00:07:02 and thereby building up the concentration of the form that we have now

00:07:07 so that when life started, it was stuck with that form.

00:07:12 And from there on, it was unable to change.

00:07:17 Now, the isomer of one molecule has completely different chemical and physical properties?

00:07:25 No, the chemical and physical properties can be quite similar.

00:07:29 The main difference has to do with the rotation of polarized light, its optical properties.

00:07:39 In other words, D-glucose and L-glucose, as far as I know, I'm not sure of this,

00:07:44 but I believe they have about the same melting point and the same molecular weight, etc.

00:07:52 So nature produces one form over the other.

00:07:56 And if you produced the same sort of proteins in the laboratory,

00:08:00 would you get the same results that nature does?

00:08:02 Yeah. As a matter of fact, if you synthesize them by chemical methods,

00:08:09 the one important aspect of that kind of technique is to develop methodology

00:08:16 which will avoid the formation of any of the wrong isomer.

00:08:21 So people have worked for years and years and years

00:08:24 to develop very clever techniques for peptide synthesis, for example,

00:08:28 where they try to avoid any racemization.

00:08:32 So you can then build up an L-L-L-L-L chain,

00:08:37 and any introduction of a D by mistake, some chemical mistake,

00:08:42 makes that preparation unsuitable.

00:08:46 In nature, it's much easier because our proteins, for example,

00:08:50 are made by translating DNA, which is totally specific in the genes,

00:08:59 into RNA, and then RNA into protein through a complex intercellular mechanism

00:09:06 which can only use L-amino acids.

00:09:10 So the selection is there, and it's a question of lock and key.

00:09:18 Are there implications that this has towards, I guess, down the road,

00:09:27 producing substances for mass production?

00:09:30 Are there specific applications of this isomer phenomenon?

00:09:37 Well, yeah. I mean, this is a common problem in industry,

00:09:41 the food business, biotechnology.

00:09:47 For example, some people don't tolerate lactose in milk.

00:09:55 So you can remove it enzymatically.

00:09:57 You can take an enzyme which splits lactose into glucose and galactose,

00:10:03 but it only attacks the correct optical isomer of lactose.

00:10:11 And you know that that's what's in the milk originally.

00:10:15 So you take this enzyme, which has been designed by nature

00:10:18 over the millions of years to do that specific job,

00:10:21 and you can pass milk through a column with this enzyme stuck to it,

00:10:25 and it will chew up the lactose.

00:10:28 That's one very sort of dumb example,

00:10:31 but that kind of basic approach is used in all sorts of systems,

00:10:38 especially in biomedicine where you're worried about the reaction of people

00:10:41 to drugs and so on.

00:10:48 You know, I think in the food stuff business,

00:10:50 I can't think of anything particular other than the milk at the moment.

00:10:54 I was wondering if there was some application in the vitamins industry.

00:10:56 It would seem to me that, I mean, I would see, for example, L-lycine sold at the shop.

00:11:00 L-lycine?

00:11:01 Yeah.

00:11:02 Well, L-lycine is an amino acid, right?

00:11:05 There are a number of amino acids that are deficient in certain,

00:11:10 corn, for example, is deficient in.

00:11:13 I think it's lysine, threonine, and tryptophan, I believe.

00:11:17 So for areas like some parts of Central America

00:11:20 where people live very much on corn products,

00:11:25 they can become deficient in these essential amino acids

00:11:32 so that since they grow corn and nothing else,

00:11:36 they have to be fed either wheat that's been shipped in

00:11:40 or else they can have their diet supplemented with synthetic amino acids

00:11:47 or more usually from these amino acids that have been isolated from seaweed and so on.

00:11:53 The Japanese have an enormous industry for the separation of pure amino acids in the L-form

00:11:59 from seaweed kelp, which I think they use kelp.

00:12:05 And the optical property theme goes through the whole thing.

00:12:12 Is there, I guess I want to go back to the idea of how they can form.

00:12:18 I'm not sure that I understand exactly, for example, the glucose.

00:12:23 How they can form two different, you would think that there would be some sort of determinant.

00:12:29 Well, in natural processes, you don't get the two forms.

00:12:33 You get only one because of this enzymatic specificity.

00:12:37 If you synthesize a molecule that has, let's say, a carbon atom

00:12:43 that's about to be changed by attaching something to one of its four bonds,

00:12:50 if you attach it to bond one instead of two, you get the D-form instead of the L-form.

00:12:58 So that synthetically, as I mentioned before,

00:13:02 you have to work out methods that always attach to two and not to one.

00:13:10 In nature, there's no problem that's taken care of.

00:13:13 It is a big problem in isolation of substances for nutritional use and other uses,

00:13:23 the problem of avoiding conditions that might possibly cause

00:13:28 vasectomization of optically active substances.

00:13:31 Have there been any examples of those problems?

00:13:34 Well, there is this one that I've been told about,

00:13:37 the thalidomide situation where abnormal babies were born to mothers who had taken this drug,

00:13:45 induced, as I recollect, by there being some of the wrong isomer present in the drug.

00:13:53 I can't back that up with facts, but there are, I'm sure, more examples of that kind of thing.

00:13:59 You were talking earlier about the size of the molecule sometimes has,

00:14:07 if it's, for example, if it's going to block an isomer,

00:14:10 I mean, if an isomer comes in and it's going to block the enzyme,

00:14:13 what's the relationship between the size of the molecule and the isomer,

00:14:18 and its, I guess, ability to trick the enzyme?

00:14:20 I see.

00:14:21 Well, most of the reactions of the sort you're talking about

00:14:28 have to do with a large molecule attacking a small molecule.

00:14:32 And the large molecules are so formed, and in spite of their size,

00:14:37 they really are only significant in terms of a small patch on their surface,

00:14:42 which forms spontaneously from the sequence of the amino acids.

00:14:47 And that small patch recognizes this small molecule.

00:14:50 The rest is window dressing, which you need to keep the small site in its proper form.

00:14:56 So if a mixture comes along of DNL and this thing recognizes only one of them,

00:15:01 or sometimes both, and then there are problems of inhibition and so on.

00:15:08 So that's the story of how DNL glucose could be separated,

00:15:18 or perhaps you might say purified, by dumping in some yeast.

00:15:23 The yeast would destroy the natural form and leave the unnatural form,

00:15:29 which we don't want anyway,

00:15:31 but at least it showed that you could take a biological system

00:15:34 and purify a mixture of isomers.

00:15:38 What happens exactly to the enzyme once it's been triggered?

00:15:41 In that case?

00:15:42 Yeah, if this doesn't work.

00:15:43 Oh, the enzyme then does its job and releases the products,

00:15:48 and then it waits for another one.

00:15:50 The enzymes are there more or less forever,

00:15:54 and they go on catalyzing this particular reaction ad infinitum.

00:16:01 But not any benefit to the natural system that it's supposed to be benefiting?

00:16:05 Well, it does the job it's supposed to do.

00:16:08 It takes, for example, let's say an energy-yielding compound

00:16:15 that can be oxidized, and oxidizes it,

00:16:20 and then can transfer electrons to an acceptor molecule, that sort of thing.

00:16:26 Each enzyme represents a step in a chain usually,

00:16:30 and the whole thing is extremely complex, some systems more than others.

00:16:36 In the gut, where we digest proteins with pepsin, for example, in the stomach,

00:16:42 the job is relatively simple.

00:16:44 It's all a question of splitting peptide bonds,

00:16:48 and pepsin just splits whatever it sees that it likes.

00:16:52 Whereas with, say, something like carbohydrate metabolism, sugar metabolism,

00:16:57 there are dozens of steps, one following each other in a specific series,

00:17:03 that have to be obeyed.

00:17:05 So depending on the biological system you're interested in,

00:17:11 the complexity can really increase to very large degrees.

00:17:17 So in summary, the relationship between, for example,

00:17:21 the wrong isomer and the natural process is what?

00:17:27 Well, the relationship is that the wrong isomer,

00:17:30 A, is not likely to be found in nature,

00:17:33 and if it is, it's avoided, or it's not used.

00:17:38 In general, I think the world is pretty much built up of one form of almost everything,

00:17:46 just by the natural selection of both the animal and the substance,

00:17:51 throughout a lot of time,

00:17:55 so that at the moment things are working smoothly.

00:18:00 We're out of time.

00:18:08 Oh, yeah, go ahead and check, Chad.

00:18:10 All right.

00:18:13 So I was going to ask you about what's going on next door.

00:18:17 Yeah, like I said, I was just getting into the calcium release.

00:18:24 Well, what I'm going to do is homogenate the actual old sites

00:18:29 and then fractionate them, you know, centripetally,

00:18:32 and work on the actual endoplasmic reticulum vesicles directly,

00:18:38 which is something that really hasn't been done yet.

00:18:41 And what I basically want to do is try to draw some sort of correlation

00:18:45 between the sarcoplasmic reticulum, which has a questionable calcium,

00:18:52 so try to draw a connection between that and the endoplasmic reticulum.

00:18:56 You're involving energy production in the endoplasmic reticulum.

00:19:00 Yeah, yeah.

00:19:04 For instance, caffeine is known to induce the calcium release from the sarcoplasmic reticulum,

00:19:10 and Busa himself has taken an entire old site and just added caffeine topically.

00:19:21 Okay.

00:19:41 Dan, let's start off first and talk about nitrogen in the atmosphere

00:19:46 and why there's so much of it.

00:19:49 How much is out there and why is there so much of it?

00:19:53 There is a lot of nitrogen, as everyone is fairly familiar, in the atmosphere.

00:19:58 It composes of almost 80% of the atmosphere.

00:20:03 I think it's basically there because it is a very inert molecule.

00:20:08 The bonds between the—it's called dinitrogen.

00:20:12 Frequently people nowadays refer to it as dinitrogen rather than just nitrogen

00:20:16 because it is two molecules with a triple bond between them,

00:20:20 and these is a very stable bond.

00:20:23 Because of it being so stable, it is very difficult to break that bond,

00:20:28 and there are very few processes that can do so.

00:20:31 In the atmosphere, things like lightning, ozonation,

00:20:37 cause a certain amount of rupture of the triple bond,

00:20:41 and this is converted into—by rainfall, then, this reaches the Earth's surface.

00:20:48 Most of the nitrogen that is converted from the atmosphere into a fixed form, though,

00:20:54 occurs by biological nitrogen fixation.

00:20:58 Biological nitrogen fixation roughly occurs—

00:21:02 Biological nitrogen fixation is roughly two-thirds of all the nitrogen fixed in the Earth.

00:21:09 Man-made nitrogen, which is principally in the form of fertilizers,

00:21:13 accounts for about 20% of all the nitrogen fixed,

00:21:17 and the remaining is a biological process, as we were talking about,

00:21:21 such as lightning in the atmosphere.

00:21:24 But I think basically it's there because it is an inert molecule,

00:21:27 and it is in equilibrium with the trapped nitrogen that's in the Earth's crust,

00:21:32 where it's trapped in rocks and deposits of all sorts, and in the ocean, of course.

00:21:38 What sort of—I mean, if lightning breaks apart a little bit of this nitrogen,

00:21:42 what breaks apart most of this tremendously strongly bonded element?

00:21:48 It is the process of biological nitrogen fixation.

00:21:53 This is a very unique process developed sometime in the course of evolution

00:22:01 in which a few microorganisms have developed an enzyme we call nitrogenase.

00:22:07 It's a protein and a very interesting molecule which binds nitrogen

00:22:13 and can reduce it to ammonia at low temperature and low pressures.

00:22:20 This is contrasted, of course, with the commercial process,

00:22:24 a chemical process called the Haber-Bosch process,

00:22:27 which is almost uniquely used commercially these days to make nitrogenous fertilizers.

00:22:34 Well, how is it that one little one-cell organism, micro-organism,

00:22:39 knew what these huge chemical factors were?

00:22:43 How is that possible?

00:22:47 That's a very good question, John.

00:22:51 One of the goals of nitrogen fixation research

00:22:55 is to learn how the molecular construction of that nitrogenase compound

00:23:04 that the bacteria has learned to make.

00:23:06 It contains an inorganic structure called an iron-molybdenum cofactor,

00:23:12 which is the target from which nitrogen is bound on the enzyme

00:23:17 and which the bacteria then reduces to ammonia.

00:23:21 This iron-molybdenum cofactor, which is an inorganic structure

00:23:25 of which we don't know the exact structure of yet,

00:23:28 has been postulated to be a model for developing a new catalyst

00:23:33 which could be used commercially for breaking this nitrogen-nitrogen triple bond

00:23:41 and using it at new commercial processes of low temperature and pressure.

00:23:51 Kind of flubbed that a little bit, didn't I?

00:23:54 Where do the little one-cell organisms live in a plant?

00:24:00 I should go back.

00:24:02 All plants need nitrogen, don't they?

00:24:05 All plants need nitrogen.

00:24:07 All living organisms need nitrogen in one form or another.

00:24:15 There are several types of microorganisms out there,

00:24:19 ones which we call free-living nitrogen fixers,

00:24:21 which live found universally all over the Earth,

00:24:26 both in the oceans and waters and soils.

00:24:29 There are the so-called blue-green algae,

00:24:32 which live also universally, primarily in aqueous environments,

00:24:41 which can reduce nitrogen.

00:24:43 But by and large, most of this nitrogen is fixed.

00:24:52 Now, for the most part...

00:24:54 Let's see, where were we?

00:24:57 Yes.

00:24:58 For the most part, the largest amount of nitrogen is fixed in plants we call legumes.

00:25:05 Legumes are examples of plants.

00:25:08 Soybeans is one of our largest commercially, agronomically important plants.

00:25:13 Other legumes are things like peas, clovers, alfalfas, cowpeas, peanuts.

00:25:20 These are all examples of legumes.

00:25:22 Legumes have learned how to utilize these nitrogen-fixing microorganisms

00:25:31 by harboring them in things we call nodules on the roots.

00:25:35 Would you like me now to go ahead and...

00:25:39 I am going to now take this plant down, as we call it, in structures.

00:25:45 We take it down by, when we're ready to study something,

00:25:48 it's on the roots of the plant.

00:25:53 This soybean plant, by the way, is about six weeks old.

00:26:00 Around behind you and around you, you can see soybeans in all states of germination and development.

00:26:07 About six weeks, the nodules should become fairly mature.

00:26:12 And if I can find a few of them here by shaking off this vermiculite,

00:26:18 which is a substrate we use to grow the plants,

00:26:22 and we water them with a plant solution,

00:26:25 so they're growing in a totally nitrogen-free environment.

00:26:29 We're incorporating no fixed nitrogen into what we fertilize them with.

00:26:38 And perhaps here you can see the nodules.

00:26:40 Do you want me to bring this closer?

00:26:44 Here we can see all the little nodule structures that have developed on the plant's roots.

00:26:50 Within these nodules are millions and millions of bacteria.

00:26:55 Bacteria that we call rhizobia.

00:26:57 In this particular case of soybeans, they're specifically designated as Rhizobium japonicum.

00:27:03 And the nodules themselves develop...

00:27:08 It's a very unique structure.

00:27:10 No structures like this are found on any other type of plants.

00:27:13 And they're developed in response to that bacteria, what we call infecting the nodule.

00:27:19 But it is a controlled, limited infection,

00:27:23 in which the plant now acts as a little house for the rhizobia.

00:27:33 For this little house that the plant develops,

00:27:39 the bacteria supply the plant with ammonia from the atmospheric nitrogen.

00:27:45 The plant, in turn, provides the bacteria with food from photosynthate, we call it.

00:27:53 It's really supplied to the nodules in the form of sucrose.

00:27:56 And there the plant further metabolizes the sucrose into other carbohydrates,

00:28:02 which the bacteria use as the source of food and energy for reducing nitrogen.

00:28:11 Now, you said that the nodules supply the plant to the bacteria.

00:28:16 They supply the plant with ammonia?

00:28:19 With ammonia.

00:28:20 What does the plant do with that?

00:28:21 The ammonia then converts the ammonia into amino acids.

00:28:26 Amino acids, of course, are the basic building block of proteins,

00:28:30 which it uses for both the vegetative growth and, of course, eventually to the seed growth,

00:28:37 which is what our product is, which we want.

00:28:41 Now, both depend on each other, correct?

00:28:46 This is a very unique combination, which is very specific.

00:28:50 It is a very specialized interaction between the bacteria and the plant.

00:28:57 There are many other factors involved also.

00:28:59 There are plant hormones, which are involved in developing this structure.

00:29:03 There are recognition factors that the plant produces from the roots,

00:29:09 which allows only certain bacteria to infect the roots in the soil.

00:29:17 In a typical, if we went out into a field where legumes had been grown before

00:29:22 and picked up one gram of soil, which would probably be about that much in my hand,

00:29:27 you would find anywhere from a thousand to a million rhizobial cells,

00:29:34 in addition to all the other types of soil bacteria.

00:29:39 Only a few of these will be the specific bacterium

00:29:42 which will cause this formation of nodules on this plant.

00:29:47 When a special bacteria comes in contact with that root,

00:29:54 and the root, in turn, secretes some recognition factors,

00:29:59 and then the rhizobia, in turn, responds to those recognition factors

00:30:03 to invade the roots of the plants,

00:30:05 and only this very special, specific symbiosis develops.

00:30:11 Now, we've talked a little bit about the strength of that nitrogen bond,

00:30:18 and here we have a bunch of little one-cell microorganisms taken apart.

00:30:22 How much energy does that plant have to supply to get the ammonium from the microorganisms?

00:30:28 That's a very good question.

00:30:33 It is one of the questions that we have to deal with

00:30:36 in considering how to improve symbioses.

00:30:39 For instance, one of the ultimate goals of scientists this day,

00:30:47 particularly in the era of what we call molecular biology and genetic engineering,

00:30:52 is to be able to incorporate the capacity for nitrogen fixation that these legumes have

00:31:00 into our other major crop plants, such as corn and wheat, oats, and so forth.

00:31:06 That is, of course, a dream, but a dream which may become a reality sooner than we might expect.

00:31:14 But one of the things that you must consider in doing that is what is the cost to the plant?

00:31:19 The plant, of course, has an infinite source of energy, which is sunlight,

00:31:25 but it can only harvest a certain amount of energy based upon the leaf area,

00:31:29 how much chlorophyll there is, the size of the plant, and so forth.

00:31:34 It can manufacture a limited amount of photosynthate, carbohydrate, for use by the bacterium,

00:31:42 because with this photosynthate, the plant has to support the vegetative parts of the plant as well, and the seed yield.

00:31:50 We currently believe that about 15 to 20 percent of all the energy that is produced by the plant

00:31:57 goes to reducing nitrogen or providing the structures which support the nitrogen fixation.

00:32:06 So this we have to take into consideration.

00:32:09 However, the interesting thing about this energy requirement is that even if we supply this plant with nitrates,

00:32:18 commercial fertilizer, it requires almost as much energy for that plant to take up nitrates from the soil

00:32:26 and to transform it and reduce it to ammonia, which it does, as it does to fix nitrogen from the atmosphere.

00:32:33 That was going to be my next question.

00:32:35 I see we're trying to build different microorganisms for different plants.

00:32:43 What's the relationship between natural nitrogen fixing and, for example, the artificial fertilizers?

00:32:52 Is there any kind of comparison that we can make there?

00:32:57 We produce a heck of a lot of fertilizer.

00:32:59 If we put this on a net, yes, I think I see the question that you're after now.

00:33:07 How much energy does it take commercially to supply this plant with nitrogen

00:33:12 versus what it is if we're supplying it biologically?

00:33:17 The commercial, on an energetic basis, if we compare the Haber-Bosch process,

00:33:23 which takes hydrogen, which it produces from natural gas, from methane,

00:33:29 and combines it in the presence of an iron catalyst to produce ammonia,

00:33:34 the process is about 30% efficient, I believe.

00:33:39 If we put this on a current basis of how much energy in the terms of oil would that represent,

00:33:47 we are currently using something like 300 million barrels of oil per year

00:33:53 in this country alone to produce nitrogen fertilizers.

00:33:59 The process in the plant is roughly the same efficiency.

00:34:02 About 30% of the energy, it's about 30% efficient.

00:34:07 But all of that energy comes from the sunlight, which is a renewable source of energy.

00:34:12 And this is the main advantage that we have in looking for biological nitrogen fixation

00:34:17 and trying to incorporate that and use that into our agronomic practices over commercial fertilization.

00:34:24 We, for instance, predict that we're going to need to double the food supply over the next 20 years.

00:34:30 Where is that energy going to come from? Where is the fertilizer going to come from?

00:34:35 To accommodate that, if it comes only from man-made nitrate fertilizers,

00:34:42 it's going to take about a quadrupling of the current capacity of nitrogen fixation.

00:34:50 So what we want to do is be able to utilize legumes much more widely and efficiently

00:34:58 in our farming practices so that we don't have to use all this commercial fertilizer.

00:35:05 There's one other interesting aspect that comes from the utilization of biological nitrogen fixation also,

00:35:12 and that is that our considerations of groundwater contamination.

00:35:18 Everybody recognizes nowadays that groundwater contamination with nitrate runoffs

00:35:25 from agricultural usage, from pesticide usage, and so forth, is a very serious problem.

00:35:32 Utilization of biological nitrogen fixed materials will greatly decrease that problem

00:35:38 of contamination of our groundwaters, particularly with nitrates.

00:35:43 I'm confused a little bit about that. What effect does nitrogen contamination have on groundwater?

00:35:51 What are the dangers there?

00:35:54 Nitrates are toxic to all animals and people, and the EPA has set certain limits

00:36:02 in which they consider safe limits for nitrates in groundwaters.

00:36:07 I don't remember what those are right now, but they recognize it as a significant source of contaminants.

00:36:16 Two questions, I want to go back.

00:36:19 How long do you want to take?

00:36:21 About three minutes.

00:36:23 Three minutes.

00:36:25 Okay.

00:36:39 All right. Let's see.

00:36:44 I want to get back about that enzyme, that magic little enzyme that nature has developed.

00:36:51 Is it the same enzyme? How many solutions to this process has nature come up with?

00:36:56 This is one of the very unique enzymes in all of nature because it is the only solution

00:37:06 that nature has come up with for biologically reducing nitrogen.

00:37:11 Whenever it was developed in the course of evolution, it has been maintained

00:37:16 throughout all these millions of years in essentially the same form in the bacterium.

00:37:21 Now it is, as the bacteria and blue-green algae have evolved,

00:37:26 they have maintained the same structures for this particular enzyme

00:37:30 so that we look and study this enzyme in the various microorganisms that contain it.

00:37:36 It's essentially the same protein molecule.

00:37:41 The studies that we do on one microorganism, we can almost translate per se

00:37:46 to that enzyme and other organisms.

00:37:49 It is truly a unique solution and a problem that nature has come up with.

00:37:54 Normally in the course of evolution, nature will change enzymes rather dramatically

00:38:02 so that in some cases there is almost no relationship between the mechanism

00:38:07 that one plant or animal uses to carry out the same process.

00:38:11 In the case of nitrogen fixation, just the opposite is true.

00:38:14 We call it being very highly conserved in nature.

00:38:18 The genes that are incorporated into all these organisms are effectively the same.

00:38:23 We could take the gene and extract it from one organism

00:38:26 and put it into another organism with the same result in protein.

00:38:32 The protein per se is...

00:38:34 Do you want me to go into this business about the structure of it a little bit?

00:38:39 It's a very interesting molecule in the sense that it's a complex molecule

00:38:44 and as we mentioned it's unique.

00:38:46 It consists of two proteins.

00:38:48 One very small protein, which is what we call an iron protein,

00:38:53 which uses iron in the protein molecule.

00:38:57 It is a reductase molecule.

00:39:00 Electrons from substrates in the cell are used to reduce this iron protein

00:39:07 and the electrons then are transmitted to a much larger molecule

00:39:11 which we call the nitrogenase enzyme per se

00:39:14 or a molybdenum iron protein.

00:39:18 The molybdenum iron protein is the actual site of binding of the dinitrogen atom

00:39:23 in which it is reduced to ammonia.

00:39:26 For that reduction to take place, three things have to happen.

00:39:30 One, the electrons have to be transmitted from the small iron protein

00:39:36 to the larger molybdenum iron protein.

00:39:41 Two, energy in the form of ATP, which has come from the plant or cell

00:39:46 or the bacterial cell, is hydrolyzed to activate the...

00:39:53 increase the bond energy so that the triple bond can be broken.

00:39:58 And three, then the protons are from water.

00:40:03 Hydrogen atoms from water come in and reduce the nitrogen molecule

00:40:08 which is then broken off from the large protein in the form of ammonia.

00:40:13 It requires a very large amount of energy because of this huge activation energy

00:40:18 that's required to rupture the very stable triple bond.

00:40:26 And this energy, as I said, was in the form of ATP

00:40:31 which the plant cell ultimately provides from its photosynthate

00:40:35 from the solar energy production.

00:40:38 So these plants are literally energy pumps that free that nitrogen?

00:40:44 Yes, you can actually think of them as solar collectors,

00:40:49 almost like a solar energy cell.

00:40:52 That's what all the chlorophyll, of course, in the leaves is there for.

00:40:56 They act as little solar cells which the light is transformed into an electrical energy

00:41:01 which is then transformed into chemical energy.

00:41:04 The plant cells convert that chemical energy to carbohydrate

00:41:08 which is then translocated through the stem to the root nodules

00:41:12 where it's used to reduce the nitrogen

00:41:15 which is then translocated back to the leaves

00:41:18 and ultimately to the grain of the plant

00:41:21 as when the grain and the pods develop on here.

00:41:24 You can see one little flower that has developed on the plant

00:41:27 which will then develop into a pod where the soybean seeds will develop.

00:41:32 Now, we talked earlier in your office, we haven't talked yet on tape,

00:41:38 about the oxygen-sensitive nature of that enzyme.

00:41:45 Whenever in the course of evolution that nitrogenase developed,

00:41:49 it almost surely developed in an anaerobic environment.

00:41:53 And we know that because it is a very oxygen-sensitive enzyme.

00:41:58 Once the biochemist extracts that enzyme from the plant cell or the bacterial cell to study it,

00:42:05 he has to rigidly exclude oxygen because it is degraded,

00:42:11 what we call stoichiometrically, by oxygen.

00:42:13 One mole of oxygen can destroy one mole of the enzyme.

00:42:20 Somehow, some microorganisms have learned to protect this enzyme in various ways.

00:42:27 There are some free-living nitrogen fixers we call azotobacters

00:42:31 that we find that are typical soil microorganisms

00:42:34 which protect it by a very high rate of respiration.

00:42:37 They use carbohydrate and oxidize it to carbon dioxide

00:42:43 to remove oxygen from the site of which nitrogen fixation takes place.

00:42:49 Blue-green algae, on the other hand, have developed a rather different mechanism.

00:42:54 They do it by compartmentalization.

00:42:56 They photosynthesize during the day and produce oxygen during the day.

00:43:01 During the night, then, the energy that is stored in the form of the carbohydrate

00:43:06 is then used to reduce nitrogen,

00:43:08 which somehow is protected in a special compartment, apparently, in the cell.

00:43:12 Although I really don't know how this is done.

00:43:16 The soybean plant and other legumes have taken a different approach to it.

00:43:20 They have chosen to build this little house, as we talked about,

00:43:26 the nodule for the bacteria to reside in.

00:43:29 Within this nodule, the plant has developed a hemoglobin type of nodule

00:43:36 which surrounds the bacteria.

00:43:38 It's very similar to human hemoglobin that you find in blood cells.

00:43:41 In blood cells, of course, the hemoglobin binds oxygen

00:43:44 and carries it to the cells where it's utilized.

00:43:49 In the case of the plant, what we call leghemoglobin, for legume hemoglobin,

00:43:56 there it is used to bind the oxygen and prevent it from getting to the site of the bacteria.

00:44:02 Thereby, the bacteria are residing in essentially an anaerobic environment

00:44:06 within the nodule cell.

00:44:10 Now, what are these plants used for?

00:44:15 These legumes, and what makes them so special?

00:44:20 You know, even before scientists knew anything about nitrogen fixation,

00:44:27 farmers, for a thousand years or more,

00:44:30 we know that even in biblical days that farmers recognized the value of legumes

00:44:36 in enriching the soil.

00:44:38 Even then, they would rotate certain legume crops,

00:44:43 I presume they were clovers or lucernes or whatever was popular in those days,

00:44:48 with other crops, and they recognized that it enriched the fields.

00:44:52 Grain crops would grow better after having legumes grown in the field the year before.

00:44:57 It was just 100 years ago this year, 1888 actually,

00:45:02 so that we're recognizing the 100th anniversary of the real discovery of nitrogen fixation,

00:45:09 of biological nitrogen fixation.

00:45:11 This occurred in Germany.

00:45:14 Two scientists named Hellriegel and Wilparth found that,

00:45:20 and convinced, more convinced the other scientists of the world

00:45:25 with very nice experimentation with legumes,

00:45:28 in which they showed that legumes had a very great advantage

00:45:32 over other grain-type crops in nitrogen-deficient soils.

00:45:37 This could only be explained by biological nitrogen fixation,

00:45:42 and it was with that discovery that the world went on then

00:45:45 to recognize biological nitrogen fixation and to study it intently.

00:45:50 Soybeans, on the other hand, are a rather modern crop.

00:45:55 While they're ancient in China,

00:45:57 most of our germplasm for soybeans has originated in China.

00:46:01 In this country, 50 years ago, soybeans were hardly grown,

00:46:05 and when they were grown, they were grown for a forage crop, just for animal feeds,

00:46:10 just the same as we would grow alfalfa today for animal feeds.

00:46:14 It is just within the last 30, 40 years that the real advantage

00:46:18 and agronomic potential of soybeans has been recognized.

00:46:22 Soybeans are very high in oil content and very high in protein content.

00:46:28 Soybeans are used to produce,

00:46:33 are the source of the world's largest source of vegetable oils.

00:46:38 They're also the world's largest source of protein-rich animal feeds,

00:46:43 which comes from crushing the remains of crushing the beans to remove the oils.

00:46:49 That protein, of course, is a direct result.

00:46:52 The high protein content is a direct result

00:46:54 of the nitrogen fixation factories in the roots of these plants.

00:46:58 There's a wonderful relationship between the efficiency of the nitrogen cycle

00:47:03 and the root change.

00:47:08 I'll have to think about that one. I'm not sure what you mean by that one.

00:47:11 If you, for example, can grow more efficient,

00:47:14 get these little rhizobia down there to crank out their nitrogen

00:47:18 and grow faster and better alfalfa and soybeans,

00:47:22 does that in turn affect, for example, cattle production?

00:47:27 Your feedstock?

00:47:30 I mean, it goes right directly into the food cycle.

00:47:33 It goes directly, yes. This is one of the things that it does.

00:47:37 It provides us with both the forage legumes and the grain legumes

00:47:43 ultimately get into the food cycle.

00:47:45 Of course, most people recognize that in Asia,

00:47:50 legumes are used much more extensively as protein sources

00:47:54 and food sources than they are in this country.

00:47:56 We use the bulk of our legume products as animal feeds.

00:48:02 In Asia, they eat and consume the products of legumes directly

00:48:10 in the forms of many, many varieties of legumes

00:48:14 that we don't even recognize in this country.

00:48:17 One of our ultimate goals, of course, is anything that we can do

00:48:21 to enhance biological nitrogen fixation to make it more efficient

00:48:25 will increase legume production and ultimately all grain production.

00:48:31 Because one of the things that we like to preach is good farming practices

00:48:35 and that involves rotating legumes, which enrich the soil with nitrogen,

00:48:40 with other grain products such as corn, wheat, oats, barley, and so forth.

00:48:45 And by this good management practices, we can decrease our consumption

00:48:50 of inorganic nitrogen fertilizers and thereby save oil.

00:48:56 Last question.

00:48:58 The notion of increasing the world's food supply

00:49:04 so tremendously in the next 20 or 30 years,

00:49:07 is there going to have to be almost a concert approach to farming

00:49:11 where we'll take man-made fertilizers, we'll take genetically enhanced fertilizers,

00:49:16 we'll take organic fertilizers?

00:49:19 Is there a concerted kind of approach?

00:49:22 John, there will have to be a concerted approach

00:49:24 because we're expecting that there will be a doubling

00:49:28 of the world's need for grains within 25 years or so.

00:49:33 Translated?

00:49:36 Let me ask the question again.

00:49:39 John, is there going to have to be a concerted approach

00:49:42 towards farming towards the use of these fertilizers and nitrogens?

00:49:48 John, within the next 25 years,

00:49:51 based upon what we expect the population growth to,

00:49:57 how we expect population growth,

00:50:00 we expect that we'll have to double the production of grain legume

00:50:04 or grain food grains within this 25-year period.

00:50:09 That can be accomplished in two ways, or more than two ways,

00:50:12 but it will have to be a concerted.

00:50:14 The best approach is a concerted approach.

00:50:17 To double our food supply of grains,

00:50:20 it will probably require a quadrupling of the need for nitrogen fertilizers

00:50:24 because nitrogen fertilizers are the limiting factor in grain production nowadays.

00:50:32 This would be a tremendous increase in costs

00:50:37 as far as our natural gas and oil resources go.

00:50:41 Everyone realizes that ultimately

00:50:44 we are going to have to conserve our fossil fuel sources.

00:50:47 The best approach, of course, is enhancing biological nitrogen fixation

00:50:52 along with the increased use of chemical fertilizers.

00:50:56 There are several different approaches to this.

00:50:59 One is the genetic approaches to enhancing biological nitrogen fixation,

00:51:05 two conservation approaches,

00:51:07 two increasing the efficiencies of fertilizer usage.

00:51:12 We roughly now, because of inefficient application practices

00:51:17 and the fact that there is the so-called biological nitrogen cycle

00:51:21 which returns nitrogen fertilizers to the atmosphere in the form of nitrogen,

00:51:27 fertilizer efficiency usage by plants is not more than 50 or 60%.

00:51:32 By good agronomic practices,

00:51:34 finding ways to control this denitrification process,

00:51:37 we should be able to increase that to at least 70%.

00:51:40 And thereby, this will give us a concerted approach.

00:51:44 So, three ways, enhancing biological nitrogen fixation,

00:51:48 increased man-made nitrogen fertilizers,

00:51:52 and three, enhanced agronomic efficient use of fertilizers

00:51:58 should be able to answer and should be able to handle

00:52:04 our increased needs for fertilizers and food production.

00:52:15 Why don't you show me how these leaves act as solar collectors

00:52:18 and where the energy goes down to the leaves.

00:52:23 John, the plant, the architecture of a plant is very important.

00:52:28 It is, what it does is acts as a solar collector for radiant energy.

00:52:33 The plant ideally will focus the broad surfaces of the leaves

00:52:38 directly to the sunlight to collect these as if they were little solar cells.

00:52:43 This, the energy is converted to electrical energy

00:52:47 which is then converted in the plant cell

00:52:49 to chemical energy which is translocated via the stems

00:52:55 to the roots of the plant where it is used as energy

00:52:59 for both root growth and to support nitrogen fixation.

00:53:04 I've got a question about those nodules.

00:53:06 Can you lift up that little plastic piece so we can see them?

00:53:10 Do they tend to grow in one area of the plant or another?

00:53:15 It seems like they're all, they cluster.

00:53:19 This is one of the interesting biological control processes.

00:53:23 The plant actually limits the number of nodules that it will,

00:53:28 that will develop on this plant.

00:53:30 As you might imagine, the whole root surface is exposed to the bacteria.

00:53:35 So why aren't there nodules down here?

00:53:38 The answer is the plant knows how many nodules

00:53:43 or how many bacteria it can support by its photosynthetic processes

00:53:48 and thereby limits the number of nodules that it will develop

00:53:52 on any particular root sort, on any particular root.

00:53:56 This particular plant developed the nodules up here,

00:53:59 what we call crown, basically crown nodules.

00:54:02 If we had allowed this plant to develop somewhat

00:54:05 before we applied the bacteria to it,

00:54:08 the nodules would have been more widely distributed

00:54:11 on other roots which were later developing.

00:54:15 But this is basically because, one,

00:54:17 the nodules developed early on the crown area of the plant

00:54:21 and two, it limits the number of nodules that will develop

00:54:25 based upon its need for nitrogen fixation.

00:54:28 And it limits it because those nodules just take too much energy to support?

00:54:31 That's correct.

00:54:32 And the plant recognizes that and will only accept as many nodules as it can support.

00:54:37 Now, you showed me a gram of soil.

00:54:42 Show me that gram of soil again.

00:54:45 If we look at, if we went out into a soil

00:54:49 in which legumes have been grown before

00:54:52 and whether they're agronomically important legume,

00:54:55 and whether they're economically...

00:54:58 That way?

00:55:00 Yeah.

00:55:02 If we went out and found a gram of soil,

00:55:05 picked up a gram of soil, any soil where legumes have been grown before,

00:55:09 a gram of soil probably would be represented about this much

00:55:12 in the palm of your hand.

00:55:14 We would find that that gram of soil contained anywhere between

00:55:17 a thousand and a hundred thousand rhizobial cells

00:55:22 in that little gram of soil.

00:55:25 And that's in addition to all the other types of soil bacteria are present as well.

00:55:31 If we broke open one of those nodules, would it be juicy or what would it be like?

00:55:37 If you remember, we talked a few minutes ago about leghemoglobin

00:55:43 and how leghemoglobin protects the bacteria and the enzyme nitrogenase from oxygen.

00:55:49 If we break open one of these nodules,

00:55:53 you'll find that it's actually red inside.

00:55:56 And this red, maybe I hope you can, I wonder if you can get that.

00:56:02 It's pretty small.

00:56:04 You want me to come closer to it or can you sit?

00:56:07 That would be interesting because that actually shows the leg...

00:56:10 Back a little more, a millimeter.

00:56:12 Okay, good.

00:56:16 Okay, good. Now that, it looks red inside.

00:56:19 As you can see, it looks red inside and this is the leghemoglobin

00:56:23 that the plant has developed to protect the enzyme nitrogenase

00:56:29 which is in their bacterial cells from oxygen.

00:56:32 It's kind of spooky that it's the same stuff as blood.