John A

John A. Frantz, MD

February 20, 2003

Addendum :…… Getting biomass ethanol from the laboratory to the highway has been slow. BC International of Dedham, Massachusetts, which plans to build a plant in Louisiana to convert sugar cane waste into fuel, is having a hard time getting $90 million to build the refinery. “It’s a combination of the economy and the fact that it’s the first of its kind,” says Vice Pres > “Bankers and investors love to say, “ Where is one of those running?’”

U. S. news and World Report , 2-17-03 , bottom of page 38

Microbiol Biotechnol (2003) 61: 1-9 describes termites in great detail. Most of the 20 or so organisms that help termites to digest cellulose have not been cultured, but some have been > The bottom line: the products of termite digestion of cellulose include acetate, methane, hydrogen, and little or no glucose. So a giant ruminant stomach substitute looks more promising than trying to imitate termites for alcohol production. However, these mind experiments have been diverting. I still like the idea of using waste instead of a food crop. In the future, when I stray from medicine and health, I will pause a little longer for more research before going public.

How are washing machines and riding lawn mowers alike?

Our daughter was riding with us to St. Louis to our grandson’s (her nephew’s) wedding. The above question occurred to me as I observed her watching a man on his riding lawn mower mowing the 5-acre (approximately) lawn of his trophy house.

When we get washing machines, most of us don’t get the full advantage of the labor saving device because we wash more stuff more often using up much of the time and effort we might have saved. Riding lawn mowers permit the same “wheel spinning” compulsion.

And when we get good roads and automobiles, many of us spend two hours per day commuting—ample time to put in quite a large garden resulting in great health improvement for the entire family.

frantzmd.info

How Termites Live on a Diet of Wood

If only wood could be converted to biofuels, there would be no need to wait a million years for the trees to be buried and become oil. Wood is indeed convertible to useful chemicals, because termites do it every day, causing $1 billion of damage every year in the United States. But to live on a diet of wood is challenging, not least because wood contains so little nitrogen. So how do termites do it?

The trick lies in a cunning triple symbiosis, a team of Japanese scientists report in Friday’s issue of Science. In the termites’ gut lives an amoeba-like microbe called a protist, and inside each protist live some 10,000 members of an obscure bacterium.

The microbes in the termites’ gut are very hard to cultivate outside their termite host and so cannot be studied in the lab. The Japanese scientists, led by Yuichi Hongoh and Moriya Ohkuma at the RIKEN Advanced Science Institute in Saitama, have cut through this problem. They extracted the protist’s bacteria directly from a termite’s gut, collected enough to analyze their DNA, and then decoded the 1,114,206 units of DNA in the bacterium’s genome.

By comparing the DNA sequence of the bacterium’s genes with other decoded genes already in public databases, the Japanese team was able to figure out what each gene did. It could then reconstruct all the biochemical reactions of which the bacterium is capable, as shown in the figure above.

They found that in the bacterium’s biochemical repertoire is the ability to convert nitrogen (shown as N2 , to the right of center in the figure) into ammonium and hydrogen. Unlike nitrogen, which is very unreactive, ammonium is easily incorporated into biochemical reactions.

www.nytimes.com

Termites can digest cellulose because

Comparative Biochemistry and Physiology Part B: Comparative Biochemistry

1. Termites and cockroaches are excellent models for studying the role of symbionts in cellulose digestion in insects: they eat cellulose in a variety of forms and may or may not have symbionts.

2. The wood-eating cockroach, Panesthia cribrata, can be maintained indefinitely, free of microorganisms, on a diet of crystalline cellulose. Under these conditions the RQ is 1, indicating that the cockroach is surviving on glucose produced by endogenous cellulase.

3. The in vitro rate at which glucose is produced from crystalline cellulose by gut extracts from P. cribrata and Nasutitermes walkeri is comparable to the in vivo production of CO2 in these insects, clearly indicating that the rate of glucose production from crystalline cellulose is sufficient for their needs.

4. In all termites and cockroaches examined, cellulase activity was found in the salivary glands and predominantly in the foregut and midgut. These regions are the normal sites of secretion of digestive enzymes and are either devoid of microorganisms (salivary glands) or have very low numbers.

5. Endogeneous cellulases from termites and cockroaches consist of multiple endo-β-1,4-glucanase (EC 3.2.1.4) and β-1,4-glucosidase (EC 3.2.1.21) components. There is no evidence that an exo-β-1,4-glucanase (cellobiohydrolase) (EC 3.2.1.91) is involved in, or needed for, the production of glucose from crystalline cellulose in termites or cockroaches as the endo-β-1,4-glucanase components are active against both crystalline cellulose and carboxymethylcellulose.

6. There is no evidence that bacteria are involved in cellulose digestion in termites and cockroaches. The cellulase associated with the fungus garden of M. michaelseni is distinct from that in the midgut; there is little indication that the fungal enzymes are acquired or needed. Lower termites such as Coptotermes lacteus have Protozoa in their hindgut which produce a cellulase(s) quite distinct from that in the foregut and midgut.

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Termites can digest cellulose because

Assignment 12: (continued)

Activities:
ACTIVITY 1. WAYS THAT ANIMALS ACQUIRE AND DIGEST FOOD
Introduction to nutrient acquisition
Herbivorous and carnivorous fish
Feeding strategies of rotifers
Termites and symbiotic relationships
Intracellular and extracellular digestion
ACTIVITY 2. DIGESTIVE SYSTEMS OF VERTEBRATE HERBIVORES VS CARNIVORES
The digestive tracts of larval and adult frogs
The digestive tracts of rabbit and cat
The digestive tract of ruminant mammals
ACTIVITY 3. TEETH OF HERBIVORES, CARNIVORES, AND OMNIVORES

Termites and symbiotic relationships

Herbivores that feed on plants ingest food that contains large amounts of cellulose. Most animals lack the digestive machinery to digest cellulose, passing it out of the body as “dietary fiber.” Very few animals have been found to produce the necessary enzymes (cellulases) for breaking down cellulose. In the few animals that have genes for cellulase, there is some debate as to whether these genes are activated and actually produce the enzyme. There are a number of animals, however, that have been demonstrated to house symbiotic micro-organisms that can digest cellulose. One example is the relationship between termites and the symbionts housed within its gut.

View the following video that shows the contents of termite guts. Note the diversity of microorganisms contained inside the gut of a termite. There are a large number of bacteria as well as the much larger, single-celled protists. Attention first focused on the unusual protists found within the termite. They are still the focus of much research because of their unusual lifestyle and morphology, but most cannot live for long without their own symbiotic bacteria. Scientists now feel that the bacteria found within both the termite gut and the protists are responsible for the digestion of cellulose. So what once was thought to be a mutualistic relationship between protists and termites is beginning to look like a relationship in which the termite may not be harmed by the protists, but may not benefit much from their presence. The termite itself may produce some of the enzymes needed to start the digestion of cellulose in its salivary glands, but there appear to be no termite enzymes that can complete cellulose digestion. So the termite’s survival depends on the symbiotic bacteria found in its gut. You will see another example of cellulose-digesting symbionts later in this exercise.

Think about the symbiotic relationship(s) between termites and microorganisms, then answer question 6 on your work sheet.

Intracellular and extracellular digestion

Intracellular and extracellular digestion are somewhat self-explanatory terms: digestion takes place inside of cells or outside of cells (typically within a digestive cavity). For digestion to take place inside a cell, food particles must first be taken into the cell. The following video shows food being taken into Paramecium multimicronucleatum, a single-celled protist, and incorporated into food vacuoles where it will be digested.

Now study the following images to view digestion taking place within the food vacuoles of Paramecium multimicronucleatum. The food source, yeast cells, have been stained with congo red, a dye that changes to a dark blue color as acid is secreted into the vacuole.

A. Individual yeast cells can be discerned within food vacuoles. B. As digestion proceeds, material in the food vacuoles becomes less distinct and color begins to change. C. After 20 minutes many of the food vacuoles appear empty and the contents of those containing material appear dark.

Extracellular digestion in animals occurs within a cavity where the food is exposed to digestive enzymes. A simple digestive cavity, with only one opening to the exterior, is found in animals of Phylum Cnidaria. The following video shows an animal from this phylum, hydra, capturing prey and moving it into the digestive cavity. Prey is captured by stinging cells concentrated in the hydra’s tentacles. Proteins from the prey stimulate the hydra to open its mouth and move the rest of the tentacles towards the captured object. The tentacles move the prey into the mouth (opening into the digestive cavity). Enzymes are secreted by specialized cells that line the cavity. These enzymes begin to break down the food into smaller particles.

Thus,extracellular digestion enables the hydra to eat relatively large prey in comparison to its size. Another type of cell lining the cavity then engulfs the small food particles. Digestion is completed in food vacuoles within these cells (intracellular digestion) and the products are transported to the rest of the body. Undigested food is passed out of the cells into the gut cavity and are expelled through the mouth.

Most animals have a “complete” digestive tract with two openings, a mouth and an anus. This allows the sequential processing of food as it passes in one direction through the digestive tube. Review the material on vertebrate digestive systems in the topic “Acquisition of Nutrients: Animals”, then examine the following images of a frog’s internal anatomy. You can assume that the function of the frog digestive organs are the same as those of mammals.

Digestion within the food vacuoles of a paramecium

Internal Anatomy of a Frog

A. Initial dissection illustrates the position of liver in the body cavity.

B. The gallbladder is located below the liver.

C. Removing the fat bodies, allows the intestines to be seen. The function of fat bodies is to store excess lipids.

D. The digestive tract of the bullfrog has been removed from the body.

Make sure that you can identify all parts of the frog digestive system and know their function. Then answer questions 7-9 on your work sheet.

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Termites’ enzyme anomaly

By John Bonner 26 March 2007

Termites rely on symbiotic bacteria to digest cellulose, so why do they digest some cellulose themselves?

Japanese researchers have discovered a previously unknown method used by termites to digest cellulose. The discovery offers a novel source of enzymes to assist in the production of biofuels, they suggest.

Primitive groups of termites break down the normally indigestible cellulose with the aid of cellulase enzymes secreted by single-celled protozoans in the termite gut. Higher termites secrete their own cellulases directly from cells in their midgut. But Gaku Tokuda and Hirofumi Watanabe from the University of the Ryukyus, Okinawa predicted that these endogenous cellulases produced by higher termites are not sufficient to meet the insect’s energy needs.

Source: © William Rafti of the William Rafti Institute.

Termites digest the cellulose in wood, causing extensive damage.

The researchers studied the gut contents of two higher termite species, Nasutitermes walkeri and N. takasagoensis. They now report in Biology Letters that cellulase enzymes are also produced by bacteria in the hindgut of these organisms.

Tokuda believes these findings will be of interest to the US Department of Energy, which is funding genomic analysis of different termite species in its search for more efficient methods of converting agricultural and forestry waste into usable energy. He points out that the termites could be a valuable source of industrial enzymes because they have adapted to live on diets involving a wide range of plant material as well as wood.

’The termite gut is the smallest bioreactor in the world,’ said Tokuda. ’We have a lot of fundamental knowledge to learn from these micro bioreactors to establish efficient biomass conversion systems.’

But termite expert David Bignell from Queen Mary College, University of London, UK, is sceptical about claims of industrial applications of termite-based products.

’This argument has been used for the last 30 years to justify all kinds of termite research but I don’t know of any commercial applications,’ said Bignell. ’We have an extensive knowledge of microbial cellulases, and most interest from biotechnologists is focused there.’

Where the Japanese study is interesting, he said, is in highlighting an evolutionary paradox: why should the termites go to the trouble of developing their own cellulases when they can be supplied more efficiently by symbiotic bacteria? Bignell suspects that cellulases in the hindgut are there to supply the energy needs of symbiotic bacteria, not the host.

’In wood feeders the limiting factor for growth is nitrogen, not carbon, because wood contains very little nitrogen. So the bacterial community of the termite hindgut contains bacteria that fix nitrogen from the atmosphere, and nitrogen fixation is energetically expensive. Any cellulose processing going on there may just be supporting the N-fixing bacteria.’

References

G Tokuda and H Watanabe, Biol Lett, 2007, DOI: 10.1098/rsbl.2007.0073

www.chemistryworld.com

Symbiosis of Termites and the Microbes in their Gut: Digestion of Lignocellulose

Lower Termites versus Higher Termites

There is a distinction between lower termites and higher termites, mentioned throughout many studies of termite guts. Lower termites have many species of bacteria along with protozoa, while higher termites usually just have the bacteria and a more elaborate anatomy while lacking the protozoa. (3) Both higher and lower termites have microbes and enzymes in their hindgut, and this is therefore where the most symbiosis occurs. (Fig. 1) Bacteria and archaea are present in both types of termites, but cellulolytic flagellates are only found in lower termites. (8) The evolution of these flagellates has provided the termites with the ability to digest wood, and experiments have demonstrated that without these protists, lower termites starve. The dominant microbiota in lower termite hindguts are spirochaetes. (8) Higher termites do not have the protozoa that lower termites have in their hindguts that make cellulose digestion possible, so higher termites have evolved with different gut components and diets. (3) Some higher termites have a symbiosis with a fungus, Termitomyces, and Bacteroidetes and Firmicutes which thrive with a fungus-rich diet. Other higher termites that have cellulose-rich diets, have many spirochaetes and Fibrobacteres in their gut, as well as an anatomically evolved gut that has increased compartmentalization in the hindgut and increased alkalinity (more basic pH level) in anterior compartments. (8,9) It is possible that this high alkalinity could be the key to the mystery of how higher termites handle the degradation of lignin. (Figure 2)

Cellulase Activity

Termites degrade lignocellulose with the combination of their own “mechanical and enzymatic contributions” and the catalytic contribution of their symbiotic microbes. (8) Many studies on termite gut cellulose digestion have been conducted using carboxymethyl-cellulose as the substrate to measure cellulase activity in termites, but carboxymethyl-cellulose can be hydrolyzed in ways that aren’t specific to digestion in the gut, such as activity in salivary glands. Therefore, Tokuda et. al looked at the digestion of crystalline cellulose in termites and compared the different types of cellulase activity. (4) Their investigation found that most cellulose digestion occurs in the hindgut for termites that have flagellates, and the midgut for termites without the presence of flagellates in their gut. They also describe evidence of hingdgut bacteria containing ‘cellulosome’ complexes, explaining how they digest the food substrates with their cell wall by surrounding the food substrates. (4)

In lower termites, metabolic fermentation processes are credited to the flagellates, but similar to lignin degradation, less is known about the fermentation processes of higher termites. (9) The termite gut, in general, is not a simple anoxic environment, as it actually has a complex structure of different microenivronments. (5) As opposed to the bovine rumen and human colon, which only have anaerobic bacteria, the termite gut has the presence of oxygen and this effects the processes that occur. (6) For most lower and all higher termites, there is an accumulation of hydrogen in the hindgut paunches, suggesting it is a key metabolite. (8) Most of this hydrogen is used in homoacetogenesis and methanogenesis. (8) Part of what makes certain termite guts so interesting is the preference the bacteria have for acetogenesis over methanogenesis. (3) Termites that have fungi in their gut and feed on soil seem to perform methanogenesis more often than acetogenesis, but the higher termites that just feed on wood are the ones that prefer acetogenesis. (6) From what has been studied so far, formate is formed in the hindguts of many termites, which then either accumulates, oxidizes to carbon dioxide, or reduced to acetate. (8)

Lignocellulose Digestion

Lignocellulose digestion requires efficient cellulases and glycoside hydrolases to degrade the the cellulose and hemicellulose of the cell wall, but additionally a mechanism to handle the lignin barrier. This is accomplished through the combined effort of the host termite and its symbionts. (8) Both higher and lower termites have the enzymes needed for the first stage of the TCA cycle, but they lack an enzyme capable to convert pyruvate to acetyl CoA or acetate. (3) Hydrolysis of cellulose begins with endoglucanases released by the host: in lower termites the endoglucanases are secreted by the salivary glands, while they are secreted in the midgut epithelium in higher termites. (8) The flagellates in lower termites produce numerous exoglucanases, exoglucanases, B-glucosidases, and numerous other glycoside hydrolases. In higher termites, there are also many cellulases, xylanases and other glycoside hydrolases. Burnum et al. used metaproteomics to examine the hindgut of Nasutitermes corniger, a higher termite, and found almost a quarter of the 886 proteins they identified were enzymes and 36 were glycoside hydrolases. (7) Their results suggest these enzymes were important to the symbiotic relationship between the hindgut microbes and the termite host. He et al. studied Nasutitermes corniger as well as Amitermes wheeleri with the comparison of metaproteomic and metatranscriptomic analysis. (Fig. 3) Both N. corniger and A. wheeleri are higher termites, but they have different diets and habitats. (9) He et al. found different abundances of certain bacteria in the guts of the two termites, catering to their varied diets – cow dung for A. wheeleri and wood for N. corniger. In both, however, many glycoside hydrolases were found (similar to the observations of Burnum et al.) to assist in cellulose degradation. He et al. notes that there were no lignin degradation genes identified in either species’ hindguts. (9)

Further Research

It is still unclear how higher termites deal with lignin but He et al. suggest it could be the host’s own components, such as higher alkalinity in a certain part of the gut, or possibly unidentified microbes in a more aerobic component of the gut. This hole in the understanding of lignin degradation has not stopped people from researching termite gut symbiosis and how this could be used to convert lignified plant cell walls into soluble sugars. Brune et al. suggest that more knowledge on lignocellulose digestion in termites could inspire a more efficient production of biofuels in the future (8). Sethi et al. looked at three specific GHF7 (glucoside hydrolase) cellulases in the termite gut using cDNA sequences, PCR, and cellobiohydrolase activity assays. They found different levels of activity (GHF7-3 being the most active) which could be important for pursuing cellulases for biofuel production. (10)

References

(2) Hongoh, Yuichi. “Toward the Functional Analysis of Uncultivable, Symbiotic Microorganisms in the Termite Gut.” Cellular and Molecular Life Sciences 68, no. 8 (April 2011): 1311–1325. doi:10.1007/s00018-011-0648-z.

(3) Breznak, Ja, and A. Brune. “Role of Microorganisms in the Digestion of Lignocellulose by Termites.” Annual Review of Entomology 39 (1994): 453–487. doi:10.1146/annurev.en.39.010194.002321.

(4) Tokuda, G., N. Lo, and H. Watanabe. “Marked Variations in Patterns of Cellulase Activity Against Crystalline- Vs. Carboxymethyl-cellulose in the Digestive Systems of Diverse, Wood-feeding Termites.” Physiological Entomology 30, no. 4 (December 2005): 372–380. doi:10.1111/j.1365-3032.2005.00473.x.

(5) Ohkuma, M. “Termite Symbiotic Systems: Efficient Bio-recycling of Lignocellulose.” Applied Microbiology and Biotechnology 61, no. 1 (March 2003): 1–9. doi:10.1007/s00253-002-1189-z.

(6) Brune, A. “Termite Guts: The World’s Smallest Bioreactors.” Trends in Biotechnology 16, no. 1 (January 1998): 16–21. doi:10.1016/S0167-7799(97)01151-7.

(7) Burnum KE, Callister SJ, Nicora CD, Purvine SO, Hugenholtz P, Warnecke F, Scheffrahn RH, Smith RD, Lipton MS (2011) Proteome insights into the symbiotic relationship between a captive colony of Nasutitermes corniger and its hindgut microbiome. ISME J 5:161–164.

(8) Brune, Andreas. “Symbiotic Digestion of Lignocellulose in Termite Guts.” Nature Reviews. Microbiology 12, no. 3 (March 2014): 168–80. doi:10.1038/nrmicro3182.

(9) He, Shaomei, Natalia Ivanova, Edward Kirton, Martin Allgaier, Claudia Bergin, Rudolf H Scheffrahn, Nikos C Kyrpides, Falk Warnecke, Susannah G Tringe, and Philip Hugenholtz. “Comparative Metagenomic and Metatranscriptomic Analysis of Hindgut Paunch Microbiota in Wood- and Dung-feeding Higher Termites.” PloS One 8, no. 4 (2013): e61126. doi:10.1371/journal.pone.0061126.

(10) Sethi, Amit, Elena S Kovaleva, Jeffrey M Slack, Susan Brown, George W Buchman, and Michael E Scharf. “A GHF7 Cellulase from the Protist Symbiont Community of Reticulitermes Flavipes Enables More Efficient Lignocellulose Processing by Host Enzymes.” Archives of Insect Biochemistry and Physiology 84, no. 4 (December 2013): 175–93. doi:10.1002/arch.21135.

(11) Scharf, Michael E, Zachary J Karl, Amit Sethi, and Drion G Boucias. “Multiple Levels of Synergistic Collaboration in Termite Lignocellulose Digestion.” PloS One 6, no. 7 (2011): e21709. doi:10.1371/journal.pone.0021709.

Edited by Diana McDonnell, a student of Nora Sullivan in BIOL168L (Microbiology) in The Keck Science Department of the Claremont Colleges Spring 2014.

microbewiki.kenyon.edu

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