Microbial Physiology 6 2013

MICR540 !Microbial Physiology 6 ! Johnson 2013

Microbial Physiology 6 Engineering Microbes for the Future

Introduction

Dental caries is a slow demineralization of the tooth caused by a loss of hydroxypatite crystals. It has been around since the beginning of recorded history. Before the Iron Age, between 2-4% of teeth showed decay. During the Iron Age and through the Roman period, caries increased to about 10%. Some children from the middle-ages had extensive caries, likely caused by paciﬁers made of honeycomb wrapped in linen. Caries increased dramatically toward the end of the seventeenth century and, except for a reprieve during world war II, have continued to rise in the Western world until a decade ago. Underdeveloped countries from the 50's and 60's showed a marked change from low to high caries after exposure to a Western diet.

Historically, there have been some hot-spots for caries, particularly in the United States. In the Northeast, molasses was a major export and swallowed copiously during the 1700's. A visitor to this region wrote:

The inhabitants of this province (Massachusetts) are formed by symmetry, handsome and have delicate complexions . . . but, both sexes have universally and even proverbially bad teeth, which must probably be occasioned by their eating so much molasses.

By the Revolutionary War most 18 year olds from this region failed the physical exam for military duty. It required that the person had two matching front teeth for biting open a container of powder! Military records highlight dental deterioration during the 20th century. During World War II, 8.8% of recruits did not meet the requirement of 6 opposing teeth. A recent by the World Health Organization estimates that 5 billion people world-wide (>70% of the world's population) presently suffer from tooth decay.

Why are bacteria implicated? Several lines of evidence point to bacteria as the major cause of caries: i) germ-free animals do not develop caries; ii) bacteria can demineralize the enamel in vitro or in vivo and iii) bacteria are found within the enamel and dentin of carious lesions.

There is good data indicating that Streptococcus mutans is the major offending organism. This organism sticks to the enamel, sets up shop and begins to ferment any carbohydrate it can use to lactic acid, bringing the pH below the critical level of 5.5, where the enamel begins to dissolve. S. mutans itself is quite stable at this pH. If one has more than 1 million S.. mutans per ml of saliva, one is at high risk for dental caries.

Why is sucrose special?

History has shown qualitatively, that sucrose is an important factor in forming caries. More recent scientiﬁc studies within the last hundred years have shown a direct correlation between sucrose and caries, long before S. mutans was identiﬁed as an agent. The Table

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shows and example of such a correlation in a rat model. In vitro experiments showed that S. mutans can ferment starch, lactose, glucose and mannitol to pH 5.0 and below. How then does sucrose serve so well as a cariogenic substrate for S. mutans? The answer is that S. mutans can rapidly convert sucrose into a sticky long-chain polysaccharide capsule. This capsule acts as a glue to stick to the tooth surface and plaque mass. It is also a fuel source during when nutrients are not available

Sucrose is a disaccharide made of glucose and fructose. The link between them is a dihemiacetal bond, which has a high free energy of hydrolysis. The more negative the values are in the Table, the greater the energy is when the bonds are split. S. mutans can use this high energy to make the sticky glucose polymer dextran (-2 kcal/mol) or the fructose polymer levan (-4.6 kcal/mol).

Susceptibility

There is little question that susceptibility is not equal in all individuals. There are host factors that favor as well as inhibit the development of caries. For example, it is obvious that teeth that contain deep pits and ﬁssures, such as those often present in molars, can be particularly susceptible to plaque formation. There are also salivary components.

Active salivary components

Antibacterial proteins Saliva contains a host of antibacterial proteins

Buffers Saliva has phosphate and bicarbonate buffers, but bicarbonate is more important. As salivary ﬂow increases, bicarbonate increases but phosphate decreases. Bicarbonate also has a dissociation constant in the range where plaque acids reacts rapidly by losing CO2.

Pellicle components Teeth have a naturally occurring non-bacterial ﬁlm from salivary proteins that regularly forms on teeth. It may be brushed away, but it will reform within minutes. This serves as a base for plaque formation. The salivary pellicle may also increase the resistance of the enamel to acid.

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Immunoglobulins Saliva contains secretory IgA, which is resistant to oral proteolytic enzymes. In at least one study, salivary IgA was higher in children with no caries than those with caries. Saliva also contains IgG and IgM from the gingival sulcular ﬂuid. These immunoglobulins could potentially intercept bacteria and decrease their plaque formation. Several antigenic determinants on S. mutans have been used as baits to develop immunity (see section on vaccines)

Antimicrobial agents and treatments

Anions Anions include Fluoride and Sodium Lauryl sulfate (detergent).

Cations: Cations such as Zn++ or cationic detergents such as Chlorhexidine are attracted to the bacterial cell walls because of the substances' positive charge and the negative charge of the bacterial cell wall. Gram positive bacteria are more sensitive to cations since they are more negatively charged. S.mutans are Gram positive bacteria and are therefore very sensitive to cations.

Non-ionic Agents There are two types of phenol like substances. One is triclosan which is a non- charged agent and the other is Listerine™ which is a combination of the phenol-related essential oils thymol and eucalyptol. (Thymol is also a constituent in the chlorhexidine varnish Cervitec). Triclosan is normally not the only antimicrobial agent in different vehicles. To augment the efﬁcacy the surface coating copolymer, polyvinylmethyl ether and maleic acid, commercially known as Gantrez™, is added.

Enzymes A glucanhydrolase has been isolated by some Korean chemical engineers that prevents adherence to S. mutans to glass, even in the presence of sucrose.

Speciﬁc Treatments and Prevention

Fluoride Fluoride has been proven to reduce the incidence of caries. It has been added to drinking water, to table salt, milk and toothpastes, as well as in a variety of mouthwashes, gels and varnishes for topical use, and in tablets. Fluoride is also found naturally in water, tea and ﬁsh.

Fluoride is thought to increase the resistance of tooth enamel to acid by replacing the hydroxyl group of hydroxyapatite, producing ﬂuorapatite.

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If fluoride is present in the saliva, it will promote remineralization of fresh enamel lesions. Since fluorapatite is more resistant to demineralization by acid, fluoride helps reduce enamel solubility when in the mineral structure.

High concentrations of topical ﬂuorides also reduce the acid production of cariogenic bacteria. Fluoride inhibits an enzyme required for transporting sucrose into the cell.

Fluoride does not generally cause a dramatic decrease in salivary S. mutans levels after “normal” use. On the other hand, lower numbers of cavities occur, decreasing the number of sites for easy colonization. Populations with optimal ﬂuoride concentration in the drinking water are somewhat less colonized by S. mutans, compared to similar populations with less ﬂuoride in the water.

Chlorhexidine Chlorhexidine, originally used as a skin disinfectant, has remarkable anti-plaque activity. An early study found that no plaque accumulated in volunteers who rinsed their mouths with a 0.2% rinse twice a day for 3 weeks. There was an 85-90% reduction in salivary ﬂora and tooth surfaces remained bacteria free.

Chlorhexidine is highly positively charged and sticks to oral surfaces, where it is slowly released in an active form. It is this feature, and not superior antibacterial properties, which make it effective. Its positive charges also help it stick to bacteria for the kill. Chlorhexidine has an unpleasant, bitter taste that may interfere with taste perception. It also stains teeth yellow-brown over time, but this can be removed by dental polishing.

Brushing and Flossing

While brushing and flossing do decrease smooth surface caries, studies have shown that they are not effective against fissure and pit caries. The reason is these that pits cannot be effectively reached and cleaned with a brush. There is also some evidence that S. mutans can survive on a brush for some period after brushing. Whether reinfection can occur with this brush and whether flossing can spread the organism from one tooth to another is a subject of debate.

Regular profession debridgement A study in the early >70s in Sweden showed that biweekly dental treatment including debridement and topical fluoride could markedly decrease dental caries. Other studies that included professional cleaning with a non-fluoridated paste were less successful. One reason for this is the rapid rate of recolonization on the tooth surface. Thus, fluorideʼs antimicrobial and remineralization features may be required to enhance the debridement to change plaque flora from cariogenic to non-cariogenic.

Sealants

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Sealants are a breakthrough in preventive treatment of caries prone areas such as pits and ﬁssures. Leakage is minimal, and microbes trapped below the sealant don=t have enough nutrients to survive. Therefore, sealants can be placed above an incipient lesion and it wonʼt progress. Bacteria can populate the sealant, but their acid products cannot penetrate it.

Eliminating sugar

When a campaign was launched for lowering sugar intake to prevent dental caries in the middle of this century, it had little impact. Although people believed the message, little was done to curb consumption. "When head and hedonism clash it is easy to predict the winner" (Irwin D. Mandel, Director, Center for Clinical Research in Dentistry, Columbia University).

Sugar substitutes A sugar substitute will not be used if the taste differs much from sucrose. Many artiﬁcial sweeteners are too sweet, and must be diluted out. Some carbohydrates lack the characteristic sweetness of sucrose. The Table below summarizes some properties of some sugar substitutes. Table of sugar substitutes.

Compound Sweetnessa Chemical Nature Regulatory Status

Monelin 2000 Small protein Saccharin 300 to 500 salt Approvedb Aspartmae 180 to 200 dipeptide Limited approval Cyclamate 40 salt Disapproved Fructose 1.3 hexose Food High Fructose 1 to 1.2 enzyme treated Approved Corn Syrup starch Sucrose 1 disaccharide Food Xylitol 1 penitol Special dietaryc Glucose 0.7 Hexose Food Mannitol .07 hexitol Food additive Sorbitol .5 hexitol Food additive Lycasin 7 hydrogenated starch Maltitol disaccharide alcohol Corn Syrup 0.1 to .7 starch hydrolysate Food 1. Arranged in order of relative potency with sucrose and xylitol considered as ideal, i.e. a score of one. 2. Approved status under investigation. 3. Will not permit widespread usage of xylitol as a food additive.

Another problem with some sugar substitutes, is that they can also be fermented by S. mutans. Remember that sucrose is necessary for S. mutans to synthesize the sticky glucans for colonization, but once colonized, it will happily ferment many other carbohydrates! In the Table below, the fermentation end products produced by S. mutans given different carbohydrate substrates is shown. The Table (right panel) also gives relative values of acid produced by various oral microbes when fermenting different sugars.

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Acid Production in vitro from Sucrose Substitutes by Variouis Streptococcal and Lactobacilli Species

Sucrose Substitute Fermentation Pathways of Carbohydrates and Polyols Glucosea Lycasin7 Mannitol Sorbitol Xylitol

Compound ChemicalHydrogen Fermentation End Products Species Number of Strains Exhibiting Strong Positive Formula Carbon Fermentation 4 molecules lactic Carbohydrate (CH20)n 2 4(Ch3CH2OHCOOH) Streptococcal sp. S. avium 1 1 0 1 1 Sucrose C12H24O12 22 moles lactic 2(Ch3CH2OHCOOH) S. faecalis 26 26 0 26 0

S. faecium 4 4 0 0 0 Glucose C6H12O6 22 moles lactic 2(Ch3CH2OHCOOH) S. milleri 17 1 0 0 0 S. mitior 14 0 0 3 0 Fructose C6H12O6 2(1 mole lactic (Ch3CH2OHCOOH) S. mitans 15 0 0 13 0 S. salivarius 15 0 0 0 0 Polyols (CH2O)n +2H (1 mole formica HCOOH S. sanguis 26 10 0 12 0 C6H24O6 (1 mole ethanol Lactobacilli sp. Sorbitol 2.33 CH3CH2OH C6H23O6 L. casei 39 33 30 38 0 L. fementum 1 0 0 0 0 Mannitol 2.33 (1 mole acetic CH3COOH (1 mole ethanol L. salivarius 3 3 0 3 1 C5H12O5 CH3CH2OH b Xylitol 2.4 (1 mole formica HCOOH

Fig. (Loesche) Of several fermentable sugar substitutes, xylitol is the only one that does not alter the plaque pH

Gum

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Gum chewing stimulates salivary ﬂow which washes tooth surfaces and raises the plaque pH. However, one study showed a dramatic difference in caries between groups chewing xylitol sweetened gum and sucrose sweetened gum. The xylitol gum was non-cariogenic (80% reduction in caries score!), while the sucrose gum was cariogenic. Thus, the small amount of sucrose in the gum acted as a nutrient pulse that selected for mutans. The plaque from these individuals contained different parameters as well. The Finnish government has promulgated the use of xylitol containing chewing gum. A recent survey of Finnish Teens revealed that approximately 45% of the males and 70% of females chewed xylitol gum on a daily basis (12). Less than 1% chewed sugar sweetened gum.

In another Finnish study, mothers who chewed xylitol gum 3 times a day had lower S. mutans transmission to their child ( 9.7%) than those treated with chlorhexidine (28.6%) or ﬂuoride varnish (48.5%).

Protective foods

Some elements in foods help protect against mutans. Inorganic and organic phosphate such as phytate (present in unreﬁned cereals) absorbs to the enamel surface, making it less soluble in acid. These are in large part removed during the milling process of cereal and ﬂour products. Other protective foods negate that pH drop if taken after a sucrose drink. These include peanuts, cheese, boiled eggs and rye crisp bread. Cacao (present in chocolate) inhibits 2 of the 3 glucosyltransferases in S. sobrinus.

S. mutans inhibitor isolated from licorice

Wenyuan Shi, Ph.D., a microbiologist at the UCLA School of Dentistry, created orange- ﬂavored sugarless lollipops infused with a molecule isolated from licorice that prevents the growth of S. mutans. Wenyuan ran some 50,000 experiments on 2,000 Chinese herbs, and found many anti-mutans peptides. The most active molecule was a Glycyrrhizol A, which is a pterocarpene compound, and is the agent that is in the lollipop.

(Glycyrrhizol A)

Importance of salivary ﬂow

Patients receiving radiation treatment in the head and neck develop rampant caries. The salivary ﬂow rate can drop from 1.3 ml/min (pre-radiation) to 0.2 ml/min (a 90% drop). They dry mouth cannot adequately dissolve foods and the patient usually changes eating habits to a cariogenic diet. Lesions are obvious within 3 months after radiotherapy and ﬁve new decayed surfaces per post-radiation month are not uncommon.

Salivary ﬂow is important in restricting drops in plaque pH. This was demonstrated by diverting saliva from the parotid ducts and evacuating the saliva in normal (non-irradiated) individuals. The table below shows the effects of saliva on plaque pH after a sucrose rinse.

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Table

Plaque pH Minutes After Sucrose Rinse with saliva without saliva

0 6.4 6.5 5 6.1 5.7 10 6.0 5.5 15 6.3 5.5 20 6.4 5.5

Antibiotics Studies show that antibiotics can be effective against caries for a limited period of time, afterwhich resistant strains develop.

Vaccines Rats that ingest whole S. mutans produce S-IgA that correlates with lower caries. There is also some evidence that serum IgG is equally responsible for this reduction. However, antibodies to streptococcal infections can often cross react with surface antigens of the heart. This can cause a rheumatic heart or endocarditis on the heart valves. Research in this area is proceeding with caution. Several potential targets are on the surface of S. mutans are being tested for vaccine candidacy.

Exogenous antibody therapy

Ma et al. engineered plants to secrete a monoclonal antibody against an epitope on the surface S. mutans. The antibody inhibited S. mutans colonization in animals, and the studies made news headlines. However, studies in humans have apparently not been successful.

Calcium Phosphate

As illustrated above, hydroxyapatite dissociates into calcium and phosphate in an acid dependent way. Saliva contains bicarbonate to buffer the acid, and calcium and phosphate, which can move the equilibrium in the direction towards hydroxyapatite formation. It should therefore be possible to increase the concentration of calcium phosphate in the saliva by means of a calcium phosphate tablet and increase the rate of enamel formation. Unfortunately, calcium phosphate is not very soluble, and readily precipitates. Creative ways have been designed to keep the calcium and phosphate apart until they are brought together on the surface of the

8 MICR540 !Microbial Physiology 6 ! Johnson 2013 tooth. Enamelon, a toothpaste that was tested here at Loma Linda, has stripes of calcium and phosphate that are combined on the toothbrush and tooth surface. The goal is to enrich the local tooth surface in these ions to increase the chances of enamel formation. Another method used to replace hydroxyapatite is Recaldent, or "amorphous" calcium, which is added to Trident White gum. A peptide fragment of milk casein binds calcium and phosphate in separate locations within the peptide, avoiding precipitation. The peptide has been put into mints and chewing gum as a means to push the calcium and phosphate into crevices on the tooth surface. Eric Reynolds of the Dept. Dentistry at the University of Melbourne has reported 20-30% resurfacing of enamel (purposefully pre-etched with acid) within 2 weeks of chewing the gum 4 times a day for 20 minutes.

Diseases and mutans Many diseases can indirectly inﬂuence the population of mutans. A change in rate of saliva formation and composition can occur; medicine may contain fermentable carbohydrates and lower the pH directly or by affecting the saliva, radiation towards the head-neck-region can destroy the salivary glands; poor formation of the enamel due to diseases in early childhood can lead to a state that can promote caries.

Bioengineering to the rescue; Super-strain of non-pathogenic S. mutans!

JD Hillman at the University of Florida, constructed a super strain of S. mutans that i) synthesized high levels of mutacins and that ii) could not ferment carbohydrates to lactic acid. Previous attempts were unsuccessful because the mutations inhibiting lactic acid production were lethal. Hillman's group replaced lactate dehydrogenase with alcohol dehydrogenase, so that ethanol replaced lactic acid as the major product. Rats infected with this strain showed no increase in caries when fed high levels of sucrose. The super strain thrived, competing out other S. mutans strains, without affected the ﬂora of other strains. The higher the level of sucrose, the happier the strain became. The authors envisage infecting young children with the strain as a way to permanently attenuate pathogenic variants.

Bioremediation

Microbes for Degradation Processes

Microorganisms are the workhorses of the bioremediation process. The microorganisms responsible for pollutant degradation are usually bacteria but can also be fungi. Bacteria that are capable of degrading a wide range of substances are present in almost all subsurface materials. Microbes usually need not be added to the soil in a bioreactor since they are usually present in adequate amounts. The exception being when a toxic substance has removed all endemic microorganisms. To survive, microorganisms require a supply of nutrients and an electron acceptor. As you know, aerobic organisms use oxygen as the ﬁnal electron acceptor and generally use organic carbon as a carbon source. Anaerobic organisms can use sulfate, carbon dioxide or a host of other electron acceptors such as nitrates, iron, and manganese.

Nitrogen and phosphorous are the main nutrients added to reactor mixtures. A general rule of thumb for N and P loading is ﬁve parts nitrogen and one part phosphorus. Micronutrients such as Ca, Fe, Mg, Mb, and S are usually present in sufﬁcient amounts in the soil to adequately supply microbe metabolism.

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USING BIOREACTORS TO CONTROL

AIR POLLUTION Bioreactors have been used for hundreds of years to treat sewage and other odoriferous,water-borne waste. About sixty years ago, Europeans began using bioreactors to treat contaminated air (odors), particularly emissions from sewage treatment plants and rendering plants. More recently they have been used to consume pollutants from a contaminated air stream (such as a factory). The initial process used a device called a "bioﬁlter." A bioﬁlter is usually a rectangular box that contains an enclosed plenum on the bottom, a support rack above the plenum, and several feet of media (bed) on top of the support rack. See Figure.

Plenum: space where gas pressure is higher than that of the outside atmosphere

A large number of materials are used for bed media such as peat, composted yard waste, bark, coarse soil, gravel or plastic shapes. Sometimes oyster shells (for neutralizing acid build-up) and fertilizer (for macronutrients) are mixed with bed media. The support rack is perforated to allow air from the plenum to move into the bed media to contact microbes that live in the bed. The perforations also permit excess, condensed moisture to drain out of the bed to the plenum. A fan is used to collect contaminated air from a building or process. If the air is too hot, too cold, too dry, or too dirty (with suspended solids), it may be necessary to pre-treat the contaminated air stream to obtain optimum conditions before introducing it into a bioreactor. Contaminated air is duct to a plenum. As the emissions ﬂow through the bed media, the pollutants are absorbed by moisture on the bed media and come into contact with microbes. Microbes reduce pollutant concentrations by consuming and metabolizing pollutants. During the digestion process, enzymes in the microbes convert compounds into energy, CO2 and water. Material that is indigestible is left over and becomes residue.

In-Ground Bioﬁlters are commonly used for odor control around sewage treatment plants.

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Photograph of four Bioﬁlters being installed in Arlington, TX at Central Regional Wastewater System Plant.

Pollution in water table

Several bacteria have been isolated that can metabolize toxic substances. For example, Desulﬁtobacterium hafniense can consume and clean up waters contaminated with chlorinated compounds and solvents. These include polychlorinated biphenols. This organism has several reductive de-halogenases, and it can grow by chlororespiration on chlorinated phenolic compounds!

Superbugs may revolutionize future

The ﬁrst Geobacter species (initially designated strain GS-15) was isolated from the Potomac River, just down stream from Washington D.C. in 1987. This organism, known as Geobacter metallireducens was the ﬁrst organism found to oxidize organic compounds to carbon dioxide with iron oxides as the electron acceptor. Thus, Geobacter metallireducens gains its energy by using iron oxides (a rust-like mineral) in the same way that we humans use oxygen. Geobacter species are of primary interest because they can reduce a host of toxic metal ions, which then precipitate and come out of solution. Among these metal ions are cobalt and uranium. Uranium is a long-lived radionuclide that poses an ecological and human health hazard. The use of uranium in nuclear fuels and nuclear weapons production has created a large amount of nuclear waste, and the disposal of nuclear waste in near-surface environments remains a serious environmental issue. Uranium from radioactive waste deposits can leak into

11 MICR540 !Microbial Physiology 6 ! Johnson 2013 the groundwater system. In order to prevent further contamination of aquifers with uranium and halt the expansion of uranium contaminated ground water plumes, it is necessary to immobilize uranium in a geochemically inert form in situ. Geobacter can reduce the soluble hexavalent uranium U(VI) to tetravalent uranium U(IV), which then precipitates out as the mineral uraninite. In laboratory incubations, geobacter microbes were able to remove the soluble hexavalent (VI) uranium from the ground water.

Shewanella oneidensis Geobacter species can also metabolize petroleum contaminants, in polluted groundwater by oxidizing these compounds to harmless carbon dioxide. Perhaps the most surprising thing about this species is that it can transfer electrons directly onto the surface of electrodes. This has made it possible to design novel microbial fuel cells that efﬁciently convert waste organic matter to electricity! These bacteria sprout webs of electrical wiring that transform the soil into a geological battery. These ﬁlaments act as an electron circuit between the electron transport system the oxidizing metal surface of the electrode.

Toxic spills?

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! There are companies that specialize in bioremediation. They claim to have proprietary elixirs that are a blend of nutrients and elixirs to rapidly resolve oil spills, E. coli contamination, excess algae. I've not had a chance to investigate these data, but if you'll ﬁnd information from one of these companies is at http://www.virtualviz.com/oilspill.htm,

Alternative fuels

Ethanol is an alternative fuel to gasoline, and is at present mixed with gasoline in a form called "gasohol". Within the last 25 years, the cost of making 1 gallon of ethanol from biomass dropped from $5 per gallon to $1.20 per gallon. Problem: In the U.S., it is being converted from corn, which is food! However, the U.S produces about ~ 1.3 billion tons of low-cost biomass per year, but it isn't easy getting the energy out of it. The big problem is that energy-rich sugars in cornstalks and other crude biomass are locked up as cellulose and hemicellulose polymers. Cellulose is a polymer of six-carbon glucose units that are fairly accessible to microbial enzymes. However, hemicellulose polymers are tough to handle, because they typically contain an assortment of sugar acids as well as ﬁve- and six-carbon sugars, including xylose. The yeast Saccharomyces cerevisiae, can efﬁciently ferment glucose, but not xylose. Additionally, the biomass must undergo costly pretreatment to help tear apart the structure, such as high pressure steam, acid, etc.

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Termites to the rescue?

The termite Gut is nature's microbial bioreactor for digesting wood and making biofuels They can convert 95% of cellulose into simple sugars within 24 hours. More than 200 species of microbes make up this community, and they produce a bounty of wood-busting enzymes that could be put to work in bioreﬁneries to make ethanol from several forms of cellulosic biomass.

This diverse array of microbial capabilities that could jump-start a new biofuel industry. In addition to efﬁciently degrading cellulose into sugars, some termite-gut microbes are biochemically capable of generating other potential fuels such as hydrogen or methane. Hydrogen produced by one group of microbes is consumed by other gut microbes that create energy-producing by-products the termite can use. Investigating the termite-gut community reveals a vast collection of biological pathways that may one day be put to use for multiple energy applications.

A collaboration of researchers from the Department of Energyʼs Joint Genome Institute (DOE JGI), the California Institute of Technology, Diversa, and INBIO (National Biodiversity Institute of Costa Rica) has sequenced and analyzed microbial DNA extracted from the guts of hundreds of termites harvested from a nest in a Costa Rican rainforest. Preliminary results already have identiﬁed several novel enzymes capable of degrading cellulose into sugars, and the San Diego-based biotechnology company Diversa has used insights from this discovery to create a high-performance enzyme cocktail for processing plant biomass into biofuels. DOE JGI researchers continue to investigate other microbial communities in the guts of insects that consume different plant materials. The goal is to understand and reconstruct a diverse range of metabolic processes that could be scaled up for industrial biofuel production.

Scientists have improved fermentation processes that are used to make biofuels. Nancy Ho, a molecular biologist at LORRE, modiﬁed a strain of S. cerevisiae to ferment both glucose and xylose by inserting xylose-metabolizing genes from Pichia stipitis into the yeast genome (ASM News, October 2004, p. 442). This “Purdue yeast” boosts ethanol yields from ﬁeld residues by 40% compared to yeast strains that cannot metabolize xylose. Recently the Iogen Corporation of Ottawa, Ontario, Canada, licensed the Purdue yeast to generate ethanol from straw at a pilot plant whose output capacity is about 40 million gallons per year.

Cellulose and hemicellulose also contain several other sugars, including mannose, galactose, and arabinose. Researchers have inserted genes from ethanol-producing Zymomonas mobilis into Escherichia coli and Klebsiella oxytoca that enable them to ferment all ﬁve sugars. Although such recombinant microbes work well in the laboratory, they are not yet being used in commercial-scale processes to convert cellulosic biomass to ethanol.

Butanol may be superior

In addition to ethanol, some industrial companies are gearing up to convert biofuels to Butanol. Butanol has several advantages over ethanol, such as higher energy content, lower water absorption, better blending ability, and use in conventional combustion engines without modiﬁcation. Like ethanol, it can be produced fermentatively or petrochemically. The best- studied bacterium to perform a butanol fermentation is Clostridium acetobutylicum.

From corn to plastics (http://www.metabolix.com/)

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According to the American Plastics Council over 2 million tons of HDPE plastic per year is used for blow molded bottles, containers and other products, and are major components of the traditional petroleum-based plastics manufacturing industry. In 2006, Metabolix formed Telles™, a 50-50 joint venture with Archer Daniels Midland (ADM), to commercialize the production of Mirel™ natural plastics. Made by microbial fermentation of sugars such as corn sugar or cane sugar or vegetable oils, Mirel natural plastics are bio-based, sustainable and totally biodegradable alternatives to petroleum-based plastics that are used for many everyday products. Telles is responsible for the manufacturing, marketing and sales of Mirel natural plastics worldwide.

Use of microbes to synthesize compounds for human use:

Since 1979, when Human growth hormone was ﬁrst expressed in E. coli, bacteria have been pharmaceutical factories for synthesizing important drugs.

! One of the most important engineering feats has been the synthesis of the antimalarial drug, artimisinin, in E. coli.

Craig Venter's group takes brain storming to a new level

More recently Craig Venter, announced that his team synthesized and stitched together 580,000 base pairs of genetic code to create an entirely new and alien chromosome. Based around the Mycoplasma genitalium bacterium, the new chromosome is then implanted into a living cell and renamed as Mycoplasma laboratorium. The new "life form" is reliant on the host cell for replication and metabolism so it's not exactly entirely synthetic, but as the DNA is different, it is effectively an artiﬁcial form of life.

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Craig Venter, compares genome sequencing to "digitizing biology". Now that we have various "digital codes" it is time to take it in a new direction and design new life forms.

Some questions he has asked: Can we in fact regenerate life or even create new life? Last year, the Venter group tried to pare down the bacterial cell Mycoplasma genitalium to it's minimal components. This cell has the smallest genome of any self replicating organism. He found that they could knock out 100 of the 500 genes it possesses, but when they looked at the metabolic pathways, they concluded that the organism would not survive with all 100 genes knocked out together. That is, there is some redundancy, and some genes can help the cell survive the hit from a single gene knockout.

They wanted to synthesize the chromosome so they could vary the components and ask fundamental questions:

1. Can we chemically synthesize a chromosome? Clearly it is easy to let a bacterium do it, but can you do it on a benchtop?

2. Can chemistry permit making large molecules, and if so, can we "boot up" a chromosome from inert chemical material?

Problems:

The ability to digitize (i.e., read) the genetic code has risen exponentially, but the rate at which we can chemically synthesize DNA (e.g., primers) is some 5 orders of magnitude slower. However, Venter believes that "writing" this code will eventually catch up to reading the code, and cites the recent progress of his company. Still, speeding up synthesis isn't the only problem. It is easy to make a 30 bp DNA molecule, but the synthesis is a degenerate process, so that one accrues more errors as the length increases. A new method was created. First they synthesized DNA Phi X174 by designing pieces and correcting errors. They then put this 5,000 letter code in bacteria, the bacteria read it, made product, and the product killed the bacteria! Conclusion: the software can build itʼs own hardware.

To make 580,000 letters, they made cassettes of ~5,000 bases, but they needed to be 100% accurate. In fact, in 1995 when they sequenced M. genetalium, the accuracy of sequencing was 1 error per 10,000 letters, or 30 errors! If they had made these same 30 errors in their synthesis, they wouldnʼt have been able to boot up the cell. Part of there design was to make pieces 50 letters long and fuse them. They made unique elements to act as watermarks, to identify their homemade DNA. They fused longer and longer pieces: 5-7 kb, 24 kb, 72 kb.....they made these in abundance to sequence them and check them. However, when the size grew to 100 kB, it prevented E. coli from growing. They used homologous recombination, but realized that they were exhausting the present day tools in molecular biology.

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Lessons from Deinoccocus radiodurans

As discussed earlier, D. radiodurans can take 3 Mrads of radiation, which blows up the DNA into hundreds of tiny pieces. This organism can mend these breaks and ligate these pieces in the proper order. The Venter team used a Yeast organism that was nearly as efﬁcient as D. radiodurans recombination, They put their pieces into this yeast and watched as the pieces were put together. The genome was so big it could be seen with a regular light microscope.

The size was 580,000 letters of genetic code, over 300 million MW, It was the largest molecule EVER made. Using 10 font no spacing, it ﬁlled 140 pages!

Can this digitized information boot up another cell?

To test whether they could transplant a chromosome from 1 cell to another, the team put the new chromosome into M. genitalium. The new chromosome expressed restriction enzymes that digested the original chromosome. The new cells turned blue because of the new genes, and the new cell became a new species, with new proteins, membrane and genes. Thus, the software can change the hardware. They named the new organism Mycoplasma laboratorium. They are quick to point out that this in not genesis, but building on the shoulders of what nature has already provided.

Can microbes rescue U.S. energy needs?

From discoveries around world, we currently have a database of ~20 million genes!...these genes are the design components of the future. Venter compares the current potential of these components to the electronics business, which began with on a dozen or so components (resistors, capacitors?, etc?). The diversity electronics from such a few number of components should pale in comparison to what we can do with biological software. We are limited only by biological reality and our imagination.

Genes can be rapidly screened via cassette based constructions of millions of genomes/day. We can now use combinatorial genomics to have robots make a million chromosomes a day! Venter's groups are working to optimize these 20 million genes to produce octane for fuel, pharmaceuticals or vaccines. With small team, we do more molecular biology than all past research in the last 20 years.

What about ethanol? Many are calling the rush to make ethanol a bad experiment. But we now have microbes that can convert sugars to octane or butanol. The problem with using sugars is that much of the product is wasted as CO2, which is a greenhouse gas. Ideally, we would use CO2 as feedstock, and design cells to make fuels from this. Methanoccus jannaschi lives in geothermal vents under anaerobic conditions. It can take CO2 and with hydrogen as the energy source convert it to methane. No sugars are necessary. We are only limited by the concentration of CO2 in the air. We could get

17 MICR540 !Microbial Physiology 6 ! Johnson 2013

around that by concentrating it, or using industrial smoke stacks to trap CO2 and pipe it into fermenters. The methane could be used for gas heating, motors and more.

With methanococcus, every bit of carbon is derived from CO2. In plants, CO2 use is not that efﬁcient, but in algae, it is much more efﬁcient,. Venter's team is engineering algae to synthesize octane using sunlight and CO2. In this way, algae could be used for energy generation during the day, and Methanococcus could be used to generate energy (as methane) at night. Currently, the timetable is to design cells to make new fuels in about 18 months.

In short the goals of the Venter team are to increase understanding of life, replace petrol chemical industry, create replace, become a major source of energy, enhance georemdiation and drive antibiotic and vaccine discovery and production. Some daunting numbers: If we had microreﬁneries that produce 20,000 liters of octane a day, we would need 1 million of those to match what;s used in oil equivalents. Du pont spent 10 years and 100 million dollars to change just 16 different genes in E. coli going from sugar to propanediol! Venter's team is using computer design to be more efﬁcient.

Venter claims he knows what genes he wants for synthesis. These number about 50 genes, But for each of these they have 10 to 100,000 different varieties to choose from to look for best combos.

What about using combinatorial methods to make other important molecules?

The Venter institute apparently hired the "antibiotic" teams that were layed off from Ely Lilly after this manufacturer halted it's antibiotic program. They are also using these combinatorial methods to synthesize new antibiotics, antivirals and vaccines.