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What nutrients are best suited for growing E.Coli

What nutrients are best suited for growing E.Coli


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I am looking to grow E.Coli (In a nutrient agar dish) to be used in an E.Coli lawn and was wondering what specific nutrients should be used to ensure the E.Coli grows optimally? Any answers or links to relative resources are greatly appreciated!


Optimal is a funny thing; it depends upon what you want. The purpose of the bacteria, is probably the most important aspect when considering the nutrients. There are many recipes capable of growing E. coli. What does optimal or best mean to you and why? Is selectivity or differentiation a factor? How important is cost? Are you aiming for industrial level production or just testing. Do you have a bioreactor? Or are you looking for something low tech… maybe from food-mart?

For what it's worth, you might find minimal salts media and solid state fermentation interesting.

Different strains like different nutrients, of course. So, phenotypical tests are sometimes helpful when figuring what an undescribed strain likes (and doesn't like).

That being said… I can offer a little insight here.

"+" for growth/utilization; "-" for weak or no growth/utilization:

E. coli E. coli inactive** E. coli 0157:H7 Pectin - - - Dextrin ++ ++ - Sucrose - - +++ Glycerol +++ +++ +++ Stachyose - - - D-Fructose ++ ++ ++ D-Maltose + ++ ++ D-Raffinose + - +++ α-D-Glucose + + + α-D-Lactose + - ++ D-Trehalose ++ +++ +++ D-Galactose ++ +++ +++ L-Fucose +++ +++ +++ L-Rhamnose +++ - ++ D-Sorbitol +++ + - D-Mannitol + ++ ++ Methyl Pyruvate +++ +++ +++ Tween 40 - - -

Gelatin - - - L-Alanine +++ +++ +++ L-Arginine + - - L-Histidine - - - D-Serine +++ +++ - L-Serine +++ +++ +++ D-Aspartic Acid - - - L-Aspartic Acid +++ +++ +++

D-Glucoronic Acid +++ +++ +++

Citric Acid - - - L-Malic Acid +++ +++ +++ Acetic Acid +++ +++ +++ Acetoacetic Acid + + - L-Lactic Acid + - - Mucic Acid ++ - +++ D-Malic Acid + +++ +++ Propionic Acid ++ ++ +++ Formic Acid + - +

D-Salicin - - - Lithium Cloride +++ ++ ++ Na Butyrate +++ +++ +++ pH 5 +++ +++ +++ 8% NaCl ++ + - D-Serine (high) ++ + - Potassium Tellurate + + +++ Na Bromate +/- - ++

E. coli inactive** is lactose-negative, non-motile- often misidentified as Shigella


As suggested by Chris, classical LB medium should be fine. There is a reason why it has been used for the last 65 years. Some people supplement it with extra sucrose or sodium chloride, but I think these are mere customs rather than experimentally-proven improvement.

If you want to re-create LB from pure components, read about minimal media. They are a mixture of glucose, ammonium salts, microelements and vitamins. A rather long list that I won't post here can be found at http://structuralbiology.uchc.edu/protocols/pdfs/nmr_sample_preparation.pdf


Intra-colony channels in E. coli function as a nutrient uptake system

The ability of microorganisms to grow as aggregated assemblages has been known for many years, however their structure has remained largely unexplored across multiple spatial scales. The development of the Mesolens, an optical system which uniquely allows simultaneous imaging of individual bacteria over a 36 mm 2 field of view, has enabled the study of mature Escherichia coli macro-colony biofilm architecture like never before. The Mesolens enabled the discovery of intra-colony channels on the order of 10 μm in diameter, that are integral to E. coli macro-colony biofilms and form as an emergent property of biofilm growth. These channels have a characteristic structure and re-form after total mechanical disaggregation of the colony. We demonstrate that the channels are able to transport particles and play a role in the acquisition of and distribution of nutrients through the biofilm. These channels potentially offer a new route for the delivery of dispersal agents for antimicrobial drugs to biofilms, ultimately lowering their impact on public health and industry.


Types of Nutrient Feeding Programs - Pros and Cons

As discussed in previous posts there are 12 essential plant nutrients and fertilizing with them will help to optimize growth. For large scale agriculture there's the assumption that there are already nutrients contained in the soil and so a sample is sent off to a lab for analysis and the fertilizer is applied based upon what nutrients are deficient in the soil. For the production of high value crops it is often better to start out with "a blank canvas" having next to no nutrients and then being able to add them artificially, thereby controlling when and how much is given of a particular element.

For any of these systems there is always the potential have less than optimal levels of a particular nutrient. Also a more likely scenario is having too much of a nutrient which is wasteful.

By using all natural substances it is assumed that there a healthy soil will produce a healthy plant and in turn those consuming the crop will also be healthier.

  • Less wasteful as it's a good way to recycle food and yard wastes through composts.
  • Will produce healthier soils which has long term benefits. On the other hand large scale operations depend on tillage for weed control which contributes to soil erosion.
  • Some nutrients will be locked out being unavailable to the plant. Phosphorus is by far the biggest problem here as it can take years or decades to become absorbable by the plant.
  • Uses animal waste as a major component. This leads to possible contamination by E. coli and other pathogens in food products.

Please note that for the terminology of the following types is what it is know by locally by the author and there may be other types of names in other locations.

One Part System

This consists one component used for the whole vegetative cycle and a second used for the whole flowering cycle.

  • Nutrient levels can not be customized between weeks.
  • Contain calcium together with phosphorus and sulfur. They are not typically compatible and blending them together is a specialized process, which drives up the production cost which translates to a more expensive product for the consumer.

Two Part System

This consists of a Grow and a Flower part together with a "Common" which is usually calcium nitrate. While not as widely used now, they were popular in the past.

Three Part System

It consists of a Grow, a Bloom, and a Micro component. The Micro containing calcium nitrate and the micronutrients. It is the most common type of feeding program used in the industry.

  • More involved as the rates are always changing and thereby have a greater likelihood of user error.

Four Part System

Consists of two parts used in conjunction in vegetative growth and two parts used in conjunction during flowering.

  • Generally more a more expensive option than using a three part.
  • Not designed for a varied feeding regiment but rather the parts are used in a one to one ratio with simplicity in mind.

For anything in life one takes out what they put in and this is certainly true when it comes to growing plants. For someone who is inexperienced or just wants to grow casually casually a simple feeding program will suffice, but for the grower who wants to maximize yield potential a more advanced feeding program is a must.

Bio:
Loren, the Director of Fertilizer Technology at Future Harvest, grew up on a mixed grain and cattle farm in North West Saskatchewan. He went on to study biotechnology and worked in agrosciences in Saskatoon for several years before moving on to Future Harvest and the hydroponic plant food industry. Starting off in fertilizer production, his focus is now on fertilizer formulations and regulatory affairs. His areas of expertise include: agronomy, analytical chemistry, plant tissue culture, plant breeding, molecular biology, and plant nutrition. Outside of work, Loren collects vintage concert T-shirts and is an amateur craft brewer specializing in historical and lesser known styles of beer.


What nutrients are best suited for growing E.Coli - Biology


Introduction

This Application Report is part of a series documenting culture growth in the BioFlo 110. With appropriate vessels and control modules, the BioFlo 110 can be used to efficiently grow mammalian cells, plant cells, insect cells, yeast, and bacteria.

For this report, a standard 7.5L BioFlo 110 Advanced Fermentation Kit, NBS Catalog Number M1273-1125 was first used for an E. coli fed-batch fermentation.

Next, a BioFlo 110 Gas-Mix Controller, M1273-3104, was added, and the fermentation repeated with oxygen supplementation of the sparge gas. This second culture, described in the APPENDIX, achieved a very high dry cell weight of 57.2 g/L. Neither run was fully optimized, but the descriptions of procedures and materials as well as the data discussion will be useful to operators of similar fermentors.

Vessel
The BioFlo 110 Advanced Fermentation Kit used for this work was equipped with a heatblanketed 7.5 L fermentation vessel with nominal 5.7 L working volume. All BioFlo 110 fermentation vessels are configured with a 4-baffle stainless-steel insert, dual Rushton agitation impellers, and a high-speed, direct-drive agitation system with mechanical face-seal. Dissolved oxygen and pH probes (Mettler- Toledo) are also included with these fermentors. We expect similar results from BioFlo 110 fermentation vessels from 1 through 14 L in volume, and in both heat blanket and water jacket configurations.

Control System
The four control modules included with the Advanced Fermentation Kit were used for the first run. (See Chart)

Small Items
Liquid addition bottles (3), cables, tubing, clamps and other small items were as supplied with the BioFlo 110 fermentor.


Materials and Methods

Inoculum
The inoculum was prepared using LB broth at a 25-g/L concentration as the shake flask medium. The inoculum was cultivated overnight at 28C on a rotary shaker (NBS model G25) at 240 rpm. OD 600 nm was 5.50 at the time of inoculation. Inoculum volume was 5% of the 5 L working volume, or 250 ml.

Medium
Four and one-half liters of E. coli K12 medium was prepared and poured into the vessel for a 5L run. One-half liter of vessel volume was reserved for components, including inoculum, to be added after sterilization.

Initial Medium Composition:
Potassium phosphate monobasic (anhydrous) . . .2 g/L
Potassium phosphate dibasic (anhydrous) . . . . . .3 g/L
Ammonium phosphate dibasic (anhydrous) .. . . . 5 g/L
Tastone 900AG . . . . . . . . . . . . . . . . . . . . . . . . .5 g/L
Breox FMT 30 (International Specialty Chemicals,
Southampton, UK). (antifoam agent) . . .. . . . . . .0.35-0.4 ml/L

After autoclaving and cooling the vessel, we added:

Glucose, . . . . . . . . . . . . . . . . . . . . . . .. . . . .25 g/L
(50 ml/L of 50% sterile glucose solution)
Magnesium sulfate heptahydrate . . . . . . . . . .0.5 g/L
Thiamine . . . . . . . . . . . . . . . . . . . . . . . . . . .1 mg/L
K12 trace metals solution (1) . . . . . . . . . . . .5 ml/L

Control Setpoints
Setpoints were keyed into the controller prior to inoculation, and, except for DO which remained high, the vessel was allowed to equilibrate prior to inoculation.

Temperature . . . . . . . . . . . . . . . . . . . . .. . .37C
pH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. .7.0
Dissolved Oxygen . . . . . . . . . . . . . . . . . . .35%
Agitation . . . . . . . . . . . . . . . . . . . . .. .. . .300-1200 rpm
(responds automatically to oxygen demand)

Dissolved Oxygen (DO) Control
The DO probe was calibrated at 0%, (obtained by briefly disconnecting the cable), and at 100%, (obtained using 1,200 rpm agitation and 5L/M [1 vvm] airflow). After calibration, DO remained at approximately 100% until inoculation.

An agitation cascade was selected in the controller to maintain DO at setpoint through automatic adjustment of agitation speed. An agitation cascade increases agitation speed with increasing oxygen demand. To set up the cascade, we used the DO control display and keypad on the PCU to select:

Cascade : . . . . . . . . . .Agit
Minimum RPM : . . . ..300
Maximum RPM : . . . .1,200

Note that 1200 rpm is a high maximum, and could lead to excessive foam generation were it not for the presence of antifoam agent in the media. Note too, that smoother control occurs when the maximum rpm is limited to about 3 times the minimum rpm. Therefore, using 900 rpm as the agitation maximum would have allowed tighter control and the use of less antifoam in the media. The trade-off is reduced oxygen transfer rate, with possibly lower final cell density.

PH Control
We used liquid base to maintain pH at setpoint, relying on the acid-producing culture to lower pH if needed. The pH control parameters were:

Base . . . . . . . . . . . .. . .Sodium hydroxide, 10% solution
Pump . . . . . . . . . . .. . .Pump 1 of the 4-Pump Module
Transfer tubing . . . . . Narrow bore silicone tubing, as supplied
Vessel inlet . . . . . . . . .Triport adapter in the vessel headplate.
Probe calibration . . . .4.0 and 7.0 buffers

Controller Setup:
1) Pump 1 plugged into "BASE" power-outlet of the Power Controller
2) pH Control Selections: Multiplier = 50%
Dead-band = 0
PID values: factory defaults


Results and Discussion
The DO and agitation trend graphs reveal the fermentation history. Figure 1 shows that the DO declined rapidly to the 35% setpoint during the first hour. Figure 2 shows the early stage more clearly. Once the DO setpoint was reached, the control cascade varied agitation speed to meet increasing oxygen demand.

Agitation reached the preset limit of 1,200 rpm at Elapsed Fermentation Time (EFT) 3.75 hours. Further culture growth over the next 3/4 hour resulted in dissolved oxygen decreasing to well below setpoint.

A decline in agitation and rise in dissolved oxygen began at EFT 4.5 hours, indicating a reduced oxygen demand. This was at least partially due to exhaustion of the carbon source. One hundred grams of glucose were added (200 ml of a 50% solution) at EFT 4.75 hours, which slowed the decline in agitation but did not restore exponential growth, suggesting that other factors were limiting.

Figures 5 and 6 show increasing OD600nm and dry cell weight (DCW) from the time of inoculation through EFT 4.5-5 hours, consistent with the DO graphs. This run entered exponential growth phase upon inoculation. The short to non-existent lag phase results from the vigor of the inoculum, and the nurturing growth environment within the vessel.

The OD600nm and DCW of the culture were fairly stable over the last 3 hours of the run. The run was conducted for a total of 8 hours. A final OD of 26.0 and a final DCW of 10.3 g/L were obtained.

The pH trend graph in Figure 3 shows an initial decline from a slightly high value towards the 7.0 setpoint. The slight undershoot to 6.95 is normal control behavior.


Enhancements
1. DO deprivation over an extended time resulted in a loss of culture vigor. Oxygen supplementation of the sparge was investigated and described in the appendix.

2. Implementing an automated feed program through BioCommand Plus software, NBS Catalog Number M1291-0000, would have accomplished a more timely addition of nutrient, possibly increasing the final cell density.


Conclusion
E. coli growth in the BioFlo 110 was successful. A culture density of 57.2 g/L DCW was achieved in eight hours using oxygen supplementation of the sparge gas (see APPENDIX), and 10.3g/L DCW in eight hours was obtained without oxygen supplementation. Neither run was optimized for controller set points. A slightly modified medium was used to yield a dry cell weight of 57.2 g/L. The BioFlo 110 fermentor is well suited for E. coli work.


APPENDIX:
Effect of Gas Mix Controller and Oxygen Supplementation

A second fed-batch run was performed, using the B ioFlo 110 Gas Mix Controller to demonstrate the impact of oxygen supplementation on final dry cell weight. A slightly modified media was used, and the feed schedule was increased in anticipation of the effects of oxygen supplementation.

Dissolved Oxygen (DO) Control
Cascade : . . . . .Agitation and Oxygen
The cascade first increased agitation, and then added oxygen gas as needed to maintain DO at setpoint.

(1) K12 Trace Metals Solution consists of: sodium chloride 5 g/L, zinc sulfate heptahydrate 1 g/L, manganese chloride tetrahydrate 4 g/L, ferric chloride hexahydrate 4.75 g/L, cupric sulfate pentahydrate 0.4 g/L, boric acid 0.575 g/L, sodium molybdate dihydrate 0.5 g/L and 6N sulfuric acid 12.5 ml/L. Note that the quantity of sulfuric acid can vary as required to dissolve the other components properly, the usual range is between 8 20 ml/L.

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Microbial Nutrition

To obtain energy and construct new cellular components, organisms must have a supply of raw materials or nutrients.

Nutrients are substances used in biosynthesis and energy production and therefore are required for microbial growth. This chapter describes the nutritional requirements of microorganisms, how nutrients are acquired, and the cultivation of microorganisms.

Environmental factors such as temperature, oxygen levels, and the osmotic concentration of the medium are critical in the successful cultivation of microorganisms. These topics are discussed in chapter 6 after an introduction to microbial growth.

The Common Nutrient Requirements of Microorganisms

Analysis of microbial cell composition shows that over 95% of cell dry weight is made up of a few major elements: carbon, oxygen, hydrogen, nitrogen, sulfur, phosphorus, potassium, calcium, magnesium, and iron. These are called macroelements or macronutrients because they are required by microorganisms in relatively large amounts. The first six (C, O, H, N, S, and P) are components of carbohydrates, lipids, proteins, and nucleic acids. The remaining four macroelements exist in the cell as cations and play a variety of roles.

For example, potassium (K + ) is required for activity by a number of enzymes, including some of those involved in protein synthesis. Calcium (Ca 2+ ), among other functions, contributes to the heat resistance of bacterial endospores. Magnesium (Mg 2+ ) serves as a cofactor for many enzymes, complexes with ATP, and stabilizes ribosomes and cell membranes. Iron (Fe 2+ and Fe 3+ ) is a part of cytochromes and a cofactor for enzymes and electron-carrying proteins.

All organisms, including microorganisms, require several micronutrients or trace elements besides macroelements. The micronutrients—manganese, zinc, cobalt, molybdenum, nickel, and copper—are needed by most cells. However, cells require such small amounts that contaminants in water, glassware, and regular media components often are adequate for growth. Therefore it is very difficult to demonstrate a micronutrient requirement.

In nature, micronutrients are ubiquitous and probably do not usually limit growth. Micronutrients are normally a part of enzymes and cofactors, and they aid in the catalysis of reactions and maintenance of protein structure. For example, zinc (Zn 2+ ) is present at the active site of some enzymes but is also involved in the association of regulatory and catalytic subunits in E. coli aspartate carbamoyltransferase. Manganese (Mn 2+ ) aids many enzymes catalyzing the transfer of phosphate groups. Molybdenum (Mo 2+ ) is required for nitrogen fixation, and cobalt (Co 2+ ) is a component of vitamin B12.

Besides the common macro-elements and trace elements, microorganisms may have particular requirements that reflect the special nature of their morphology or environment. Diatoms (see figure 26.6c,d) need silicic acid (H4SiO4) to construct their beautiful cell walls of silica [(SiO2)n]. Although most bacteria do not require large amounts of sodium, many bacteria growing in saline lakes and oceans depend on the presence of high concentrations of sodium ion (Na + ).

Finally, it must be emphasized that microorganisms require a balanced mixture of nutrients. If an essential nutrient is in short supply, microbial growth will be limited regardless of the concentrations of other nutrients.

Requirements for Carbon, Hydrogen, and Oxygen

The requirements for carbon, hydrogen, and oxygen often are satisfied together. Carbon is needed for the skeleton or backbone of all organic molecules, and molecules serving as carbon sources normally also contribute both oxygen and hydrogen atoms. They are the source of all three elements. Because these organic nutrients are almost always reduced and have electrons that they can donate to other molecules, they also can serve as energy sources.

Indeed, the more reduced organic molecules are, the higher their energy content (e.g., lipids have a higher energy content than carbohydrates).

This is because, as we shall see later, electron transfers release energy when the electrons move from reduced donors with more negative reduction potentials to oxidized electron acceptors with more positive potentials. Thus carbon sources frequently also serve as energy sources, although they don’t have to.

One important carbon source that does not supply hydrogen or energy is carbon dioxide (CO2). This is because CO2 is oxidized and lacks hydrogen. Probably all microorganisms can fix CO2—that is, reduce it and incorporate it into organic molecules.

However, by definition, only autotrophs can use CO2 as their sole or principal source of carbon. Many microorganisms are autotrophic, and most of these carry out photosynthesis and use light as their energy source. Some autotrophs oxidize inorganic molecules and derive energy from electron transfers.

The reduction of CO2 is a very energy-expensive process. Thus many microorganisms cannot use CO2 as their sole carbon source but must rely on the presence of more reduced, complex molecules such as glucose for a supply of carbon. Organisms that use reduced, preformed organic molecules as carbon sources are heterotrophs (these preformed molecules normally come from other organisms). As mentioned previously, most heterotrophs use reduced organic compounds as sources of both carbon and energy.

For example, the glycolytic pathway produces carbon skeletons for use in biosynthesis and also releases energy as ATP and NADH. A most remarkable nutritional characteristic of microorganisms is their extraordinary flexibility with respect to carbon sources. Laboratory experiments indicate that there is no naturally occurring organic molecule that cannot be used by some microorganism.

Actinomycetes will degrade amyl alcohol, paraffin, and even rubber. Some bacteria seem able to employ almost anything as a carbon source for example, Burkholderia cepacia can use over 100 different carbon compounds. In contrast to these bacterial omnivores, some bacteria are exceedingly fastidious and catabolize only a few carbon compounds. Cultures of methylotrophic bacteria metabolize methane, methanol, carbon monoxide, formic acid, and related one-carbon molecules. Parasitic members of the genus Leptospira use only long-chain fatty acids as their major source of carbon and energy.

It appears that in natural environments complex populations of microorganisms often will metabolize even relatively indigestible human-made substances such as pesticides. Indigestible molecules sometimes are oxidized and degraded in the presence of a growth-promoting nutrient that is metabolized at the same time, a process called co-metabolism. The products of this breakdown process can then be used as nutrients by other microorganisms.

Nutritional Types of Microorganisms

In addition to the need for carbon, hydrogen, and oxygen, all organisms require sources of energy and electrons for growth to take place.

Microorganisms can be grouped into nutritional classes based on how they satisfy all these requirements (table 5.1). We have already seen that microorganisms can be classified as either heterotrophs or autotrophs with respect to their preferred source of carbon. There are only two sources of energy available to organisms: (1) light energy, and (2) the energy derived from oxidizing organic or inorganic molecules.

Phototrophs use light as their energy source chemotrophs obtain energy from the oxidation of chemical compounds (either organic or inorganic). Microorganisms also have only two sources for electrons. Lithotrophs (i.e., “rock-eaters”) use reduced inorganic substances as their electron source, whereas organotrophs extract electrons from organic compounds.

Despite the great metabolic diversity seen in microorganisms, most may be placed in one of four nutritional classes based on their primary sources of carbon, energy, and electrons (table 5.2).

The large majority of microorganisms thus far studied are either photolithotrophic autotrophs or chemoorganotrophic heterotrophs.

Photolithotrophic autotrophs (often called photoautotrophs or photolithoautotrophs) use light energy and have CO2 as their carbon source. Eucaryotic algae and cyanobacteria employ water as the electron donor and release oxygen. Purple and green sulfur bacteria cannot oxidize water but extract electrons from inorganic donors like hydrogen, hydrogen sulfide, and elemental sulfur.

Chemoorganotrophic heterotrophs (often called chemoheterotrophs, chemoorganoheterotrophs, or even heterotrophs) use organic compounds as sources of energy, hydrogen, electrons, and carbon. Frequently the same organic nutrient will satisfy all these requirements. It should be noted that essentially all pathogenic microorganisms are chemoheterotrophs.

The other two nutritional classes have fewer microorganisms but often are very important ecologically. Some purple and green bacteria are photosynthetic and use organic matter as their electron donor and carbon source. These photoorganotrophic heterotrophs (photoorganoheterotrophs) are common inhabitants of polluted lakes and streams. Some of these bacteria also can grow as photoautotrophs with molecular hydrogen as an electron donor. The fourth group, the chemolithotrophic autotrophs (chemolithoautotrophs), oxidizes reduced inorganic compounds such as iron, nitrogen, or sulfur molecules to derive both energy and electrons for biosynthesis. Carbon dioxide is the carbon source. A few chemolithotrophs can derive their carbon from organic sources and thus are heterotrophic.

Chemolithotrophs contribute greatly to the chemical transformations of elements (e.g., the conversion of ammonia to nitrate or sulfur to sulfate) that continually occur in the ecosystem.

Although a particular species usually belongs in only one of the four nutritional classes, some show great metabolic flexibility and alter their metabolic patterns in response to environmental changes. For example, many purple nonsulfur bacteria act as photoorganotrophic heterotrophs in the absence of oxygen but oxidize organic molecules and function chemotrophically at normal oxygen levels. When oxygen is low, photosynthesis and oxidative metabolism may function simultaneously.

Another example is provided by bacteria such as Beggiatoa that rely on inorganic energy sources and organic or sometimes CO2) carbon sources. These microbes are sometimes called mixotrophic because they combine chemolithoautotrophic and heterotrophic metabolic processes.

This sort of flexibility seems complex and confusing, yet it gives its possessor a definite advantage if environmental conditions frequently change.

Requirements for Nitrogen, Phosphorus, and Sulfur

To grow, a microorganism must be able to incorporate large quantities of nitrogen, phosphorus, and sulfur. Although these elements may be acquired from the same nutrients that supply carbon, microorganisms usually employ inorganic sources as well.

Nitrogen is needed for the synthesis of amino acids, purines, pyrimidines, some carbohydrates and lipids, enzyme cofactors, and other substances. Many microorganisms can use the nitrogen in amino acids, and ammonia often is directly incorporated through the action of such enzymes as glutamate dehydrogenase or glutamine synthetase and glutamate synthase. Most phototrophs and many non-photosynthetic microorganisms reduce nitrate to ammonia and incorporate the ammonia in assimilatory nitrate reduction. A variety of bacteria (e.g., many cyanobacteria and the symbiotic bacterium Rhizobium) can reduce and assimilate atmospheric nitrogen using the nitrogenase system.

Phosphorus is present in nucleic acids, phospholipids, nucleotides like ATP, several cofactors, some proteins, and other cell components. Almost all microorganisms use inorganic phosphate as their phosphorus source and incorporate it directly.

Low phosphate levels actually limit microbial growth in many aquatic environments. Phosphate uptake by E. coli has been intensively studied. This bacterium can use both organic and inorganic phosphate. Some organophosphates such as hexose 6-phosphates can be taken up directly by transport proteins.

Other organophosphates are often hydrolyzed in the periplasm by the enzyme alkaline phosphatase to produce inorganic phosphate, which then is transported across the plasma membrane.

When inorganic phosphate is outside the bacterium, it crosses the outer membrane by the use of a porin protein channel. One of two transport systems subsequently moves the phosphate across the plasma membrane. At high phosphate concentrations, transport probably is due to the Pit system. When phosphate concentrations are low, the PST, (phosphate-specific transport) system is more important. The PST system has higher affinity for phosphate it is an ABC transporter and uses a periplasmic binding protein.

Sulfur is needed for the synthesis of substances like the amino acids cysteine and methionine, some carbohydrates, biotin, and thiamine. Most microorganisms use sulfate as a source of sulfur and reduce it by assimilatory sulfate reduction a few require a reduced form of sulfur such as cysteine.

Growth Factors of Microorganism

Microorganisms often grow and reproduce when minerals and sources of energy, carbon, nitrogen, phosphorus, and sulfur are supplied. These organisms have the enzymes and pathways necessary to synthesize all cell components required for their wellbeing.

Many microorganisms, on the other hand, lack one or more essential enzymes. Therefore they cannot manufacture all indispensable constituents but must obtain them or their precursors from the environment. Organic compounds required because they are essential cell components or precursors of such components and cannot be synthesized by the organism are called growth factors.

There are three major classes of growth factors: (1) amino acids, (2) purines and pyrimidines, and (3) vitamins. Amino acids are needed for protein synthesis, purines and pyrimidines for nucleic acid synthesis. Vitamins are small organic molecules that usually make up all or part of enzyme cofactors, and only very small amounts sustain growth.

The functions of selected vitamins, and examples of microorganisms requiring them, are given in table 5.3. Some microorganisms require many vitamins for example, Enterococcus faecalis needs eight different vitamins for growth. Other growth factors are also seen heme (from hemoglobin or cytochromes) is required by Haemophilus influenzae, and some mycoplasmas need cholesterol.

Knowledge of the specific growth factor requirements of many microorganisms makes possible quantitative growth-response assays for a variety of substances. For example, species from the bacterial genera Lactobacillus and Streptococcus can be used in microbiological assays of most vitamins and amino acids.

The appropriate bacterium is grown in a series of culture vessels, each containing medium with an excess amount of all required components except the growth factor to be assayed. A different amount of growth factor is added to each vessel. The standard curve is prepared by plotting the growth factor quantity or concentration against the total extent of bacterial growth.

Ideally the amount of growth resulting is directly proportional to the quantity of growth factor present if the growth factor concentration doubles, the final extent of bacterial growth doubles. The quantity of the growth factor in a test sample is determined by comparing the extent of growth caused by the unknown sample with that resulting from the standards. Microbiological assays are specific, sensitive, and simple. They still are used in the assay of substances like vitamin B12 and biotin, despite advances in chemical assay techniques.

The observation that many microorganisms can synthesize large quantities of vitamins has led to their use in industry. Several water-soluble and fat-soluble vitamins are produced partly or completely using industrial fermentations. Good examples of such vitamins and the microorganisms that synthesize them are riboflavin (Clostridium, Candida, Ashbya, Eremothecium), coenzyme A (Brevibacterium), vitamin B12 (Streptomyces, Propionibacterium)

Uptake of Nutrients by the Cell

The first step in nutrient use is uptake of the required nutrients by the microbial cell. Uptake mechanisms must be specific—that is, the necessary substances, and not others, must be acquired. It does a cell no good to take in a substance that it cannot use. Since microorganisms often live in nutrient-poor habitats, they must be able to transport nutrients from dilute solutions into the cell against a concentration gradient. Finally, nutrient molecules must pass through a selectively permeable plasma membrane that will not permit the free passage of most substances. In view of the enormous variety of nutrients and the complexity of the task, it is not surprising that microorganisms make use of several different transport mechanisms.

The most important of these are facilitated diffusion, active transport, and group translocation. Eucaryotic microorganisms do not appear to employ group translocation but take up nutrients by the process of endocytosis.

Facilitated Diffusion in Cell

A few substances, such as glycerol, can cross the plasma membrane by passive diffusion. Passive diffusion, often simply called diffusion, is the process in which molecules move from a region of higher concentration to one of lower concentration because of random thermal agitation. The rate of passive diffusion is dependent on the size of the concentration gradient between a cell’s exterior and its interior (figure 5.1). A fairly large concentration gradient is required for adequate nutrient uptake by passive diffusion (i.e., the external nutrient concentration must be high), and the rate of uptake decreases as more nutrient is acquired unless it is used immediately. Very small molecules such as H2O, O2, and CO2 often move across membranes by passive diffusion. Larger molecules, ions, and polar substances do not cross membranes by passive or simple diffusion.

The rate of diffusion across selectively permeable membranes is greatly increased by using carrier proteins, sometimes called permeases, which are embedded in the plasma membrane.

Because a carrier aids the diffusion process, it is called facilitated diffusion. The rate of facilitated diffusion increases with the concentration gradient much more rapidly and at lower concentrations of the diffusing molecule than that of passive diffusion (figure 5.1). Note that the diffusion rate levels off or reaches a plateau above a specific gradient value because the carrier is saturated— that is, the carrier protein is binding and transporting as many solute molecules as possible. The resulting curve resembles an enzyme-substrate curve and is different from the linear response seen with passive diffusion.

Carrier proteins also resemble enzymes in their specificity for the substance to be transported each carrier is selective and will transport only closely related solutes. Although a carrier protein is involved, facilitated diffusion is truly diffusion. A concentration gradient spanning the membrane drives the movement of molecules, and no metabolic energy input is required. If the concentration gradient disappears, net inward movement ceases.

The gradient can be maintained by transforming the transported nutrient to another compound or by moving it to another membranous compartment in eucaryotes. Interestingly, some of these carriers are related to the major intrinsic protein of mammalian eye lenses and thus belong to the MIP family of proteins. The two most widespread MIP channels in bacteria are aquaporins that transport water and glycerol facilitators, which aid glycerol diffusion.

Although much work has been done on the mechanism of facilitated diffusion, the process is not yet understood completely.

It appears that the carrier protein complex spans the membrane (figure 5.2). After the solute molecule binds to the outside, the carrier may change conformation and release the molecule on the cell interior. The carrier would subsequently change back to its original shape and be ready to pick up another molecule. The net effect is that a lipid-insoluble molecule can enter the cell in response to its concentration gradient. Remember that the mechanism is driven by concentration gradients and therefore is reversible.

If the solute’s concentration is greater inside the cell, it will move outward. Because the cell metabolizes nutrients upon entry, influx is favored.

Facilitated diffusion does not seem to be important in prokaryotes because nutrient concentrations often are lower outside the cell so that facilitated diffusion cannot be used in uptake.

Glycerol is transported by facilitated diffusion in E. coli, Salmonella typhimurium, Pseudomonas, Bacillus, and many other bacteria.

The process is much more prominent in eucaryotic cells where it is used to transport a variety of sugars and amino acids.

Active Transport in Cell

Although facilitated diffusion carriers can efficiently move molecules to the interior when the solute concentration is higher on the outside of the cell, they cannot take up solutes that are already more concentrated within the cell (i.e., against a concentration gradient). Microorganisms often live in habitats characterized by very dilute nutrient sources, and, to flourish, they must be able to transport and concentrate these nutrients. Thus facilitated diffusion mechanisms are not always adequate, and other approaches must be used. The two most important transport processes in such situations are active transport and group translocation, both energy-dependent processes.

Active transport is the transport of solute molecules to higher concentrations, or against a concentration gradient, with the use of metabolic energy input. Because active transport involves protein carrier activity, it resembles facilitated diffusion in some ways.

The carrier proteins or permeases bind particular solutes with great specificity for the molecules transported. Similar solute molecules can compete for the same carrier protein in both facilitated diffusion and active transport. Active transport is also characterized by the carrier saturation effect at high solute concentrations (figure 5.1).

Nevertheless, active transport differs from facilitated diffusion in its use of metabolic energy and in its ability to concentrate substances. Metabolic inhibitors that block energy production will inhibit active transport but will not affect facilitated diffusion (at least for a short time).

Binding protein transport systems or ATP-binding cassette transporters (ABC transporters) are active in bacteria, archaea, and eucaryotes. Usually these transporters consist of two hydrophobic membrane-spanning domains associated on their cytoplasmic surfaces with two nucleotide-binding domains (figure 5.3). The membrane-spanning domains form a pore in the membrane and the nucleotide-binding domains bind and hydrolyze ATP to drive uptake. ABC transporters employ special substrate binding proteins, which are located in the periplasmic space of gram-negative bacteria (see figure 3.23) or are attached to membrane lipids on the external face of the gram-positive plasma membrane.

These binding proteins, which also may participate in chemotaxis, bind the molecule to be transported and then interact with the membrane transport proteins to move the solute molecule inside the cell. E. coli transports a variety of sugars (arabinose, maltose, galactose, ribose) and amino acids (glutamate, histidine, leucine) by this mechanism.

Substances entering gram-negative bacteria must pass through the outer membrane before ABC transporters and other active transport systems can take action. There are several ways in which this is accomplished. When the substance is small, a generalized porin protein such as OmpF can be used larger molecules require specialized porins.

In some cases (e.g., for uptake of iron and vitamin B12), specialized high-affinity outer membrane receptors and transporters are used. It should be noted that eucaryotic ABC transporters are sometimes of great medical importance. Some tumor cells pump drugs out using these transporters. Cystic fibrosis results from a mutation that inactivates an ABC transporter that acts as a chloride ion channel in the lungs.

Bacteria also use proton gradients generated during electron transport to drive active transport. The membrane transport proteins responsible for this process lack special periplasmic solute-binding proteins. The lactose permease of E. coli is a well-studied example. The permease is a single protein having a molecular weight of about 30,000. It transports a lactose molecule inward as a proton simultaneously enters the cell (a higher concentration of protons is maintained outside the membrane by electron transport chain activity). Such linked transport of two substances in the same direction is called symport.

Here, energy stored as a proton gradient drives solute transport. Although the mechanism of transport is not completely understood, it is thought that binding of a proton to the transport protein changes its shape and affinity for the solute to be transported. E. coli also uses proton symport to take up amino acids and organic acids like succinate and malate.

A proton gradient also can power active transport indirectly, often through the formation of a sodium ion gradient. For example, an E. coli sodium transport system pumps sodium outward in response to the inward movement of protons (figure 5.4). Such linked transport in which the transported substances move in opposite directions is termed antiport. The sodium gradient generated by this proton antiport system then drives the uptake of sugars and amino acids.

A sodium ion could attach to a carrier protein, causing it to change shape. The carrier would then bind the sugar or amino acid tightly and orient its binding sites toward the cell interior. Because of the low intracellular sodium concentration, the sodium ion would dissociate from the carrier, and the other molecule would follow. E. coli transport proteins carry the sugar melibiose and the amino acid glutamate when sodium simultaneously moves inward.

Sodium symport or cotransport also is an important process in eucaryotic cells where it is used in sugar and amino acid uptake.

ATP, rather than proton motive force, usually drives sodium transport in eucaryotic cells.

Often a microorganism has more than one transport system for each nutrient, as can be seen with E. coli. This bacterium has at least five transport systems for the sugar galactose, three systems each for the amino acids glutamate and leucine, and two potassium transport complexes. When there are several transport systems for the same substance, the systems differ in such properties as their energy source, their affinity for the solute transported, and the nature of their regulation. Presumably this diversity gives its possessor an added competitive advantage in a variable environment.

In active transport, solute molecules move across a membrane without modification. Many procaryotes also take up molecules by group translocation, a process in which a molecule is transported into the cell while being chemically altered (this can be classified as a type of energy-dependent transport because metabolic energy is used). The best-known group translocation system is the phosphoenolpyruvate: sugar phosphotransferase system (PTS). It transports a variety of sugars into procaryotic cells while phosphorylating them using phosphoenolpyruvate (PEP) as the phosphate donor.

PEP + sugar (outside) → pyruvate + sugar -- P (inside)

The PTS is quite complex. In E. coli and Salmonella typhimurium, it consists of two enzymes and a low molecular weight heat-stable protein (HPr). HPr and enzyme I (EI) are cytoplasmic.

Enzyme II (EII) is more variable in structure and often composed of three subunits or domains. EIIA (formerly called EIII) is cytoplasmic and soluble. EIIB also is hydrophilic but frequently is attached to EIIC, a hydrophobic protein that is embedded in the membrane.

A high-energy phosphate is transferred from PEP to enzyme II with the aid of enzyme I and HPr (figure 5.5). Then, a sugar molecule is phosphorylated as it is carried across the membrane by enzyme II.

Enzyme II transports only specific sugars and varies with PTS, whereas enzyme I and HPr are common to all PTSs.

PTSs are widely distributed in procaryotes. Except for some species of Bacillus that have both glycolysis and the phosphotransferase system, aerobic bacteria seem to lack PTSs. Members of the genera Escherichia, Salmonella, Staphylococcus, and other facultatively anaerobic bacteria have phosphotransferase systems some obligately anaerobic bacteria (e.g., Clostridium) also have PTSs. Many carbohydrates are transported by these systems.

E. coli takes up glucose, fructose, mannitol, sucrose, N-acetylglucosamine, cellobiose, and other carbohydrates by group translocation. Besides their role in transport, PTS proteins can act as chemoreceptors for chemotaxis.

Almost all microorganisms require iron for use in cytochromes and many enzymes. Iron uptake is made difficult by the extreme insolubility of ferric iron (Fe 3+ ) and its derivatives, which leaves little free iron available for transport. Many bacteria and fungi have overcome this difficulty by secreting siderophores [Greek for iron bearers].

Siderophores are low molecular weight molecules that are able to complex with ferric iron and supply it to the cell. These iron-transport molecules are normally either hydroxamates or phenolatescatecholates.

Ferrichrome is a hydroxamate produced by many fungi enterobactin is the catecholate formed by E. coli (figure 5.6a,b). It appears that three siderophore groups complex with iron orbitals to form a six-coordinate, octahedral complex (figure 5.6c).

Microorganisms secrete siderophores when little iron is available in the medium. Once the iron-siderophore complex has reached the cell surface, it binds to a siderophore-receptor protein.

Then the iron is either released to enter the cell directly or the whole iron-siderophore complex is transported inside by an ABC transporter. In E. coli the siderophore receptor is in the outer membrane of the cell envelope when the iron reaches the periplasmic space, it moves through the plasma membrane with the aid of the transporter. After the iron has entered the cell, it is reduced to the ferrous form (Fe 2+ ). Iron is so crucial to microorganisms that they may use more than one route of iron uptake to ensure an adequate supply.

Culture Media in Microbiology

Much of the study of microbiology depends on the ability to grow and maintain microorganisms in the laboratory, and this is possible only if suitable culture media are available. A culture medium is a solid or liquid preparation used to grow, transport, and store microorganisms. To be effective, the medium must contain all the nutrients the microorganism requires for growth.

Specialized media are essential in the isolation and identification of microorganisms, the testing of antibiotic sensitivities, water and food analysis, industrial microbiology, and other activities.

Although all microorganisms need sources of energy, carbon, nitrogen, phosphorus, sulfur, and various minerals, the precise composition of a satisfactory medium will depend on the species one is trying to cultivate because nutritional requirements vary so greatly. Knowledge of a microorganism’s normal habitat often is useful in selecting an appropriate culture medium because its nutrient requirements reflect its natural surroundings.

Frequently a medium is used to select and grow specific microorganisms or to help identify a particular species. In such cases the function of the medium also will determine its composition.

Synthetic or Defined Culture Media

Some microorganisms, particularly photolithotrophic autotrophs such as cyanobacteria and eucaryotic algae, can be grown on relatively simple media containing CO2 as a carbon source (often added as sodium carbonate or bicarbonate), nitrate or ammonia as a nitrogen source, sulfate, phosphate, and a variety of minerals (table 5.4). Such a medium in which all components are known is a defined medium or synthetic medium.

Many chemoorganotrophic heterotrophs also can be grown in defined media with glucose as a carbon source and an ammonium salt as a nitrogen source. Not all defined media are as simple as the examples in table 5.4 but may be constructed from dozens of components. Defined media are used widely in research, as it is often desirable to know what the experimental microorganism is metabolizing.

Complex Culture Media

Media that contain some ingredients of unknown chemical composition are complex media. Such media are very useful, as a single complex medium may be sufficiently rich and complete to meet the nutritional requirements of many different microorganisms.

In addition, complex media often are needed because the nutritional requirements of a particular microorganism are unknown, and thus a defined medium cannot be constructed. This is the situation with many fastidious bacteria, some of which may even require a medium containing blood or serum.

Complex media contain undefined components like peptones, meat extract, and yeast extract. Peptones are protein hydrolysates prepared by partial proteolytic digestion of meat, casein, soya meal, gelatin, and other protein sources. They serve as sources of carbon, energy, and nitrogen. Beef extract and yeast extract are aqueous extracts of lean beef and brewer’s yeast, respectively.

Beef extract contains amino acids, peptides, nucleotides, organic acids, vitamins, and minerals. Yeast extract is an excellent source of B vitamins as well as nitrogen and carbon compounds. Three commonly used complex media are (1) nutrient broth, (2) tryptic soy broth, and (3) MacConkey agar (table 5.5).

If a solid medium is needed for surface cultivation of microorganisms, liquid media can be solidified with the addition of 1.0 to 2.0% agar most commonly 1.5% is used. Agar is a sulfated polymer composed mainly of D-galactose, 3,6-anhydro-L-galactose, and D-glucuronic acid (Box 5.1). It usually is extracted from red algae (see figure 26.8). Agar is well suited as a solidifying agent because after it has been melted in boiling water, it can be cooled to about 40 to 42°C before hardening and will not melt again until the temperature rises to about 80 to 90°C. Agar is also an excellent hardening agent because most microorganisms cannot degrade it.

Other solidifying agents are sometimes employed. For example, silica gel is used to grow autotrophic bacteria on solid media in the absence of organic substances and to determine carbon sources for heterotrophic bacteria by supplementing the medium with various organic compounds.

Types of Culture Media

Culture media such as tryptic soy broth and tryptic soy agar are called general purpose media because they support the growth of many microorganisms. Blood and other special nutrients may be added to general purpose media to encourage the growth of fastidious heterotrophs. These specially fortified media (e.g., blood agar) are called enriched media.

Selective media favor the growth of particular microorganisms. Bile salts or dyes like basic fuchsin and crystal violet favor the growth of gram-negative bacteria by inhibiting the growth of gram-positive bacteria without affecting gram-negative organisms.

Endo agar, eosin methylene blue agar, and MacConkey agar (table 5.5), three media widely used for the detection of E. coli and related bacteria in water supplies and elsewhere, contain dyes that suppress gram-positive bacterial growth. MacConkey agar also contains bile salts. Bacteria also may be selected by incubation with nutrients that they specifically can use. A medium containing only cellulose as a carbon and energy source is quite effective in the isolation of cellulose-digesting bacteria. The possibilities for selection are endless, and there are dozens of special selective media in use.

Differential media are media that distinguish between different groups of bacteria and even permit tentative identification of microorganisms based on their biological characteristics.

Blood agar is both a differential medium and an enriched one. It distinguishes between hemolytic and nonhemolytic bacteria. Hemolytic bacteria (e.g., many streptococci and staphylococci isolated from throats) produce clear zones around their colonies because of red blood cell destruction. MacConkey agar is both differential and selective. Since it contains lactose and neutral red dye, lactose-fermenting colonies appear pink to red in color and are easily distinguished from colonies of non-fermenters.

Isolation of Pure Cultures

In natural habitats microorganisms usually grow in complex, mixed populations containing several species. This presents a problem for the microbiologist because a single type of microorganism cannot be studied adequately in a mixed culture. One needs a pure culture, a population of cells arising from a single cell, to characterize an individual species. Pure cultures are so important that the development of pure culture techniques by the German bacteriologist Robert Koch transformed microbiology.

Within about 20 years after the development of pure culture techniques most pathogens responsible for the major human bacterial diseases had been isolated (see Table 1.1). There are several ways to prepare pure cultures a few of the more common approaches are reviewed here.

The Spread Plate and Streak Plate Culture Method

If a mixture of cells is spread out on an agar surface so that every cell grows into a completely separate colony, a macroscopically visible growth or cluster of microorganisms on a solid medium, each colony represents a pure culture. The spread plate is an easy, direct way of achieving this result. A small volume of dilute microbial mixture containing around 30 to 300 cells is transferred to the center of an agar plate and spread evenly over the surface with a sterile bent-glass rod (figure 5.7). The dispersed cells develop into isolated colonies. Because the number of colonies should equal the number of viable organisms in the sample, spread plates can be used to count the microbial population.

Pure colonies also can be obtained from streak plates. The microbial mixture is transferred to the edge of an agar plate with an inoculating loop or swab and then streaked out over the surface in one of several patterns (figure 5.8). At some point in the process, single cells drop from the loop as it is rubbed along the agar surface and develop into separate colonies (figure 5.9). In both spread-plate and streak-plate techniques, successful isolation depends on spatial separation of single cells.

The Pour Plate Culture Method

Extensively used with bacteria and fungi, a pour plate also can yield isolated colonies. The original sample is diluted several times to reduce the microbial population sufficiently to obtain separate colonies when plating (figure 5.10). Then small volumes of several diluted samples are mixed with liquid agar that has been cooled to about 45°C, and the mixtures are poured immediately into sterile culture dishes.

Most bacteria and fungi are not killed by a brief exposure to the warm agar. After the agar has hardened, each cell is fixed in place and forms an individual colony. Plates containing between 30 and 300 colonies are counted. The total number of colonies equals the number of viable microorganisms in the diluted sample. Colonies growing on the surface also can be used to inoculate fresh medium and prepare pure cultures (Box 5.2).

The preceding techniques require the use of special culture dishes named petri dishes or plates after their inventor Julius Richard Petri, a member of Robert Koch’s laboratory Petri developed these dishes around 1887 and they immediately replaced agar-coated glass plates. They consist of two round halves, the top half overlapping the bottom (figure 5.8). Petri dishes are very easy to use, may be stacked on each other to save space, and are one of the most common items in microbiology laboratories.

Colony Morphology and Growth

Colony development on agar surfaces aids the microbiologist in identifying bacteria because individual species often form colonies of characteristic size and appearance (figure 5.11).

When a mixed population has been plated properly, it sometimes is possible to identify the desired colony based on its overall appearance and use it to obtain a pure culture. The structure of bacterial colonies also has been examined with the scanning electron microscope. The microscopic structure of colonies is often as variable as their visible appearance (figure 5.12).

In nature bacteria and many other microorganisms often grow on surfaces in biofilms. However, sometimes they do form discrete colonies. Therefore an understanding of colony growth is important, and the growth of colonies on agar has been frequently studied.

Generally the most rapid cell growth occurs at the colony edge. Growth is much slower in the center, and cell autolysis takes place in the older central portions of some colonies. These differences in growth appear due to gradients of oxygen, nutrients, and toxic products within the colony. At the colony edge, oxygen and nutrients are plentiful. The colony center, of course, is much thicker than the edge. Consequently oxygen and nutrients do not diffuse readily into the center, toxic metabolic products cannot be quickly eliminated, and growth in the colony center is slowed or stopped. Because of these environmental variations within a colony, cells on the periphery can be growing at maximum rates while cells in the center are dying.

It is obvious from the colonies pictured in figure 5.11 that bacteria growing on solid surfaces such as agar can form quite complex and intricate colony shapes. These patterns vary with nutrient availability and the hardness of the agar surface. It is not yet clear how characteristic colony patterns develop. Nutrient diffusion and availability, bacterial chemotaxis, and the presence of liquid on the surface all appear to play a role in pattern formation. Undoubtedly cell cell communication and quorum sensing is important as well. Much work will be required to understand the formation of bacterial colonies and biofilms.


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Sneaky New Bacteria on the ISS Could Build a Future on Mars

To revist this article, visit My Profile, then View saved stories.

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In mid-March, NASA researchers announced that they’d found an unknown life-form hiding aboard the International Space Station. And they were cool with that.

In fact, for an organization known for a sophisticated public communications strategy—Mars rovers don’t write their own tweets, is what I’m saying—everyone was pretty quiet about this discovery.

It’s true that the new life wasn’t, say, a xenomorphic alien with acid for blood. It was a novel species of bacteria, unknown on Earth but whose genes identified it as coming from a familiar terrestrial genus called Methylobacterium. Typically its members like to hang out amid the roots of plants, not on the walls of space stations. Still, you’d think a probably-not-but-maybe-evolved-in-space microbe would merit a little more freaking out. Yet here we are. Nobody was exactly surprised—and the reasons why could define the future of human space exploration.

As part of an ongoing research project into the microbial life of the ISS, astronauts onboard in 2015 and 2016 swabbed down various parts of the station and sent home the wipes they used. Over the next couple of years down here on Earth, a team of researchers headquartered at the Jet Propulsion Laboratory’s Biotechnology and Planetary Protection Group isolated the microbes and sequenced their genes. One species, found on a HEPA filter in the station’s life-support system, was a garden-variety (literally!) Methylobacterium rhodesianum. But three samples—from a surface near the materials research rack, a wall near the “cupola” of windows, and the astronauts' dining table—were something new. The researchers running the project named it M. ajmalii.

It wasn’t even the first time these researchers found a new bacterium in space. They’d already found a whole other unknown bacterium in that set of ISS samples—they published a paper on that in 2017. There’s a chance that these bugs are in some sense aliens, that they evolved on the station. But it’s a thin one. Odds are they hitched a ride on cargo, or on astronauts, and the microbe hunters only noticed them because they went looking. “There are chances of evolution in space, no doubt, but the space station is so young. It’s only 20 years old. Bacteria might not have evolved in that span of time,” says Kasthuri Venkateswaran, the JPL microbiologist running the project.

What’s more interesting, maybe, is figuring out which bacteria are zeroes on Earth but heroes in the rarified, closed-loop environment of a spaceship. That’s why studying the International Space Station’s microbiome—the bacteria, fungi, and viruses that thrive on board—might be critical to the safety of missions to Mars, or permanent bases on other worlds. As on Earth, human health in space will depend in part on a healthy microbiome and a good relationship with the microbiome of the vessel or shelter. “We’re able to say that novel species carried by the crew might have some characteristics to withstand the conditions there,” Venkateswaran says. “The rest might have died. These are the things that survive.”

Space is really quite unpleasant. Outside a vessel or vacuum suit, it’d be a race to see if you died first from suffocation or freeze-drying. (The high levels of hard radiation are more of a long-term deal breaker.)

So the insides of those vessels and suits have to be closed systems. The only things that come and go are cargo and astronauts. But wherever people go, they bring their ride-along microbes with them—in their guts, on their skin, in their noses and mouths. That’s true in your house, and it’s true on the ISS. But the ISS is not like your house, and not just because it recycles air and water and you can’t open the windows. The air on the ISS is drier, with higher levels of carbon dioxide. Radiation levels are higher. There’s no gravity to speak of. (“We’re used to certain kinds of microbes staying on the floor, but they don’t stay on the floor if there is no floor,” says John Rummel, a former NASA Planetary Protection Officer, responsible for keeping aliens off of Earth and Earth life off of other places.) It smells not-so-fresh inside the ISS, and because it’s full of nooks and crannies that water droplets can float into and then adhere to, thanks to surface tension, it has lots of places where microbes can hang out.

In practice, that means that the environmental microbiome of the ISS looks a lot like the microbiome of the astronauts who live there. It even changes when the crews change, according to a 2019 study. Those researchers looked at skin, nose, and gut microbes from nine astronauts who spent anywhere from a few months to, in one famous case, a year on the ISS—comparing them to before- and after-mission samples, and to samples from the station itself. “Wall samples really looked like astronaut skin. The air filters really looked like astronaut nasal microbiomes,” says Alexander Voorhies, a consultant at Booz Allen Hamilton who was lead author of the paper, back when he was a staff scientist at the J. Craig Venter Institute. “The stuff in the air was different from the stuff on regularly handled objects.”

Still, the environment of the station (and presumably a Mars mission or a Mars base) is friendlier to some bacteria and fungi than others. No one really knows which. There are hints—just hints—that microgravity and the other weird conditions inside the ISS can change gene expression in bacteria, the snapshot of the biochemical things the bugs are doing to survive. Grown on the ISS, the laboratory workhorse bacterium Escherichia coli actually got more resistant to antibiotics than under similar conditions on Earth, for example. Now, to be sure, that’s not actually evolution the changes have to become permanent and passed on to subsequent generations for that. But it’s potentially a beginning. “Give them any stress, and they’re going to evolve,” says Anushree Chatterjee, a chemical and biological engineer at the University of Colorado and one of the authors of the E. coli paper. “You find these very hardy bacteria that can survive on the inner surfaces of the International Space Station. Resources are limited. Food is limited. So they will find new ways to grow.” (Those survivors have one real advantage: There probably isn’t much trying to eat them, either.)

Up in the macrobiome, though, the ISS is demonstrably unfriendly to humans. Just living there suppresses some immune responses. Astronauts have had reactivations of Epstein-Barr and varicella-zoster viral infections—a function of a change in immune status. Astronauts have earned a reputation for underplaying medical complaints, yet ISS crew frequently report skin rashes and upper respiratory infections as their most “notable” clinical issues, according to a 2016 survey.

It’s a potentially dangerous mix: Rare bacteria acquiring new skills and astronauts less able to fend off infection. NASA used to study all this by sampling and then trying to grow whatever they caught. Genetic sequencing techniques have made the hunt even more precise, because scientists can find bugs in smaller numbers than before. Eventually, NASA hopes to fly gene-sequencing devices on the missions themselves in 2016, astronaut Kate Rubins sequenced DNA in space for the first time, and she’s actually back on the ISS right now.

The idea is to use those technologies to look for microbes that are—or have become—pathogenic. “Microbiome monitoring might be a good way to detect perturbations as a result of exposure to the Martian environment, or to a potential Martian organism,” says Andy Spry, a senior scientist at the SETI Institute and a planetary protection consultant for NASA. “That said, we have a ways to go in our understanding of monitoring microbial communities in a spacecraft and in humans before we can use such an approach.”

Venkateswaran’s team has found undiscovered bacteria from the ISS and on Earth, in space vehicle assembly rooms. The fact that those are also referred to as “clean rooms” should tell you why that’s seemingly not so great. But unlike lots of those other novel species, this new ISS Methylobacterium might actually be useful. That genus is best known for things like helping with nitrogen fixation, turning complex nitrogen sources in soil into something a plant can use as a nutrient, which means it could help food grow on another world. Plus, M. ajmalii can resist high levels of radiation, and survive when it’s totally dried out, in a sort of suspended animation. In short, this little guy is better at space travel than any human. “We want to take advantage of this and see if we can grow it in simulated lunar soil or Martian regolith,” Venkateswaran says. “It might provide nutrients. That would be good, because we cannot take soil along with us to the moon and Mars. We have to depend on the soil there.”


Helpful Bacteria Examples

Streptomyces, lactobacillis and E. coli are some examples of helpful bacteria. To know more about the species of beneficial bacteria, read on.

Streptomyces, lactobacillis and E. coli are some examples of helpful bacteria. To know more about the species of beneficial bacteria, read on.

Since childhood, we have been taught that bacteria are harmful to health. Although, this information passed from one generation to another is correct, it is incomplete as good bacteria also exist. Helpful and harmful types of bacteria are both present but very few people know about the beneficial bacteria.

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Following are some examples of good bacteria:

Lactobacillus Acidophilus

Dairy products like yogurt are prepared by exposing milk to lactobacillus acidophilus bacteria. The bacteria reside in our small intestine. Their presence in adequate amounts is very important as the body completely depends upon lactobacillus acidophilus for the manufacturing of vitamin K and other infection fighting agents.

However, when the population of lactobacillus acidophilus decreases below the normal range, the person is likely to fall prey to various infections. Taking home-made yogurt on a daily basis is the best way to keep lactobacillus acidophilus population in the normal proportion.

Streptomyces

They are referred to as ‘good bacteria’ because prescription antibiotics used for the treatment of various bacterial infections, are manufactured with the help of streptomyces. The use of streptomyces is not just limited in the synthesis of antibacterial agents. Bacteria belonging to the class of streptomyces are also utilized to make antifungal agents and other medicines such as immunosuppressants that are recommended for the treatment of autoimmune disorders.

Rhizobium

Rhizobium is a soil bacteria that does an important job of supplying ammonia to plants. It is a known fact that plant growth is not possible without ammonia. Ammonia is an excellent nutrient source for the plants. Unfortunately, the amount of ammonia present in the atmosphere is not enough for the plants to survive.

However, nitrogen and oxygen is abundantly present in the atmosphere. So, in order to fulfill the nutrient requirements of plants, rhizobium bacteria uses oxygen in the atmosphere to convert nitrogen into ammonia. This process is known as nitrogen fixation, which allows plants to grow properly. Hence, microbes belonging to the class of rhizobium, are also referred to as nitrogen-fixing bacteria.

E. coli

E. coli acronym for Escherichia coli come in the list of helpful bacteria examples. Why? Simply because it plays an important role to digest the ingested food. Basically, a considerable amount of E. coli population stays in the large intestine (colon). Colon is that part of the body where most of the digestion occurs. The bacterium E. coli releases enzymes (complex proteins) that help to promote better digestion.

The enzymes secreted by the E. coli assist to speed up digestion. Besides increasing digestion power, E. coli also contribute to manufacture two essential nutrients that include vitamin K and vitamin B12. In fact, the synthesis of vitamin K and B complex vitamins like B6 and B12 in our body is due to the presence of E. coli. Keep in mind that there are different strains of E. coli and not all provide health benefits. Some can be really nasty and exposure to these can cause intestinal problems like diarrhea.

Good Bacteria List

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Following is the list of beneficial bacteria that promote health are given below:

  • Lactobaccilus Acidophilus
  • Lactobacillus Rhamnosus
  • Bifidobacterium
  • Bacillus Coagulans
  • Lactococcus Lactis
  • Lactobacillus Reuteri
  • Escherichia Coli

These are some of the species of bacteria that have been instrumental in keeping our health in good shape. Bacteria like rhizobium ensure that the plants are not deprived of adequate nitrogen, thus creating a conducive environment for their growth and nourishment.

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Some examples of bacteria are Lactobacillus, nitrogen-fixing bacteria, Bifidobacterium, Helicobacter pylori, Staphylococcus, and Streptococcus. Read on, to know more about common bacteria and some bacterial strains that are pathogenic to&hellip

Bacteria are microscopic organisms that form a huge invisible world around us, and within us. They are infamous for their harmful effects, whereas the benefits they provide are seldom known.&hellip


Escherichia coli (E. coli)

Escherichia coli is a type of bacteria that is found in healthy intestines of animals and humans, but certain strains can harm humans who ingest it.

E. Coli

Scanning electron micrograph of Escherchia coli (E. coli).

Do you ever crave raw cookie dough? What about a rare hamburger? Although these foods may sound tempting, they can harbor a type of bacteria known as E. coli, short for Escherichia coli, a rod-shaped bacteria found in soil and water. These bacteria live in the intestines of humans and animals and are important for a healthy intestinal tract. However, certain strains of this bacteria can be harmful, and even deadly.

E. coli O157:H7 is one of those strains that, if ingested, can make humans very sick. Humans who consume this type of bacteria and become infected often have symptoms such as abdominal cramping, bloody diarrhea, and vomiting. Toxins produced by E. coli O157:H7, also known as the Shiga toxin-producing E. coli (STEC), cause these symptoms. If a news station is reporting on outbreaks of E. coli, chances are they are referring to the dangerous O157:H7 strain.

The most common way that humans become infected with E. coli is from coming into contact with feces that contains E. coli. People may also become infected with E. coli from working with animals like livestock, eating undercooked meat or raw vegetables, touching the unwashed hands of someone who has come into contact with harmful E. coli strains, or drinking contaminated water.

Other than staying hydrated, there are not many ways to treat an E. coli infection. Antibiotics are not effective at combatting the infection. Therefore, prevention is important. Washing hands thoroughly with soap, avoiding unpasteurized milk, thoroughly cooking meats, and avoiding drinking water from ponds, lakes, and public swimming pools are a few ways to avoid coming in contact with harmful strains of E. coli bacteria.

Some E. coli strains are used as indicators of contaminated water. National Geographic Explorer Ashley Murray is one scientist who studies water-related diseases and waste management. Murray works as the director of Waste Enterprisers in Ghana, a company that she founded, which owns and runs waste management businesses. Other institutes, like the National Institute of Allergy and Infectious Diseases (NIAID), are researching ways to improve detection, treatment, and prevention of E. coli.

Scanning electron micrograph of Escherchia coli (E. coli).


The Sleeping Beauty of Microbes

Deep-sea creatures could hold keys to new useful microbes for synthetic biology research and . [+] biotechnology.

There’s a chance the next transformative microbe neighbors with Thermus aquaticus. Last year, a team of researchers led by the Japan Agency for Marine-Earth Science and Technology revived 100-million-year-old microbes from deep-sea sediment. Like Taq, these microbes thrive in nutrient-poor waters with very little oxygen and, according to the research team, possess a variety of metabolic functionalities.

How did these microbes survive in dormancy since the dinosaurs? What can we learn from their ancient metabolic methods? Could these be some of the organisms that help drive synthetic biology’s next leap forward?

The pace of discovery in synthetic biology is continuing to accelerate. Researchers and entrepreneurs are being empowered to tackle our planet’s most difficult challenges through biology. But to be successful, the synthetic biology space needs to fully explore its plethora of available options — out-of-the-box tools for out-of-the-box solutions to out-of-the-box problems. Could deep-sea microbes revolutionize therapeutics? Could unique algae transform carbon capture? Could an ancient organism be our next sustainable food source? The synthetic biology community has the chance to reshape its own future as the future of our planet.

Thank you to Fiona Mischel for additional research and reporting in this article. I’m the founder of SynBioBeta, and some of the companies that I write about are sponsors of the SynBioBeta conference and weekly digest.


Watch the video: Φυλλικό οξύ οφέλη και πηγές (May 2022).