1. Bacteria (bacterium) - unicellular, procaryotic cells. The bacteria are the smallest and the most numerous of the organisms in soil. There are more than 400 named genera and an estimated 104 species. Species still unknown quite likely outnumber those already known.
2. Actinomycetes (specialized bacteria) - unicellular to filamentous, procaryotic.
3. Fungi (fungus) - mostly filamentous, eukaryotic.
4. Algae (alga) and Cyanobacteria- unicellular to multicellular photosynthetic cells. The cyanobacteria (formerly the blue-green algae) are procaryotes. All others are eukaryotic.
5. Protozoa - single-celled, eucaryotes. “Forerunners” of the animal kingdom.
6. Viruses - Viruses are not organisms in the strict sense of being cells. They are better thought of as genetic elements; particles composed of DNA or RNA generally surrounded by a protein coat. They cannot reproduce unless they gain entry into a living “host” cell to do the job for them.
|Microbial Group||Example||Size (µm)||Numbers no. g-l of soil||Biomass wet mass kg ha-l of soil|
|Viruses||Tobacco Mosaic||0.02 x 0.3||1010 - 1011|
|Bacteria||Psuedomonas||0.5 x 1.5||108 - 109||300 - 3000|
|Actinomycetes||Streptomyces||0.5 - 2.0 ‡||107 - 108||300 - 3000|
|Fungi||Mucor||8.0 ‡||105 - 106||500 - 5000|
|Algae||Chlorella||5 x 13||103 - 106||10 - 1500|
|Protozoa||Euglena||15 x 50||103 - 105||5 - 200|
|Nematodes||Pratylenchus||1,000 §||101 - 102||1 - 100|
|Earthworms||Lumbricus||100,000 §||10 - 1000|
|‡ diamter of hyphae||§ length|
This tree is derived from comparative sequencing of 16S or 18S ribosomal RNA. Note the three major domains of living organisms: the Bacteria, the Archaea, and the Eukarya. The evolutionary distance between two groups of organisms is proportional to the cumulative distance between the end of the branch and the node that joins the two groups.
Bacteria - are prokaryotes, i.e. they lack a nucleus defined by a nuclear membrane (Fig. 2). They are characteristically devoid of the intracellular organelles (nucleus, mitochondria, etc.) observed in eukaryotic organisms. One theory (the Endosymbiont Theory) holds that the mitochondria as well as chloroplasts of higher organisms were derived from bacteria or cyanobacteria that entered other cells and became obligate intracellular symbiontsclass="right".
Common shapes of bacterial cells:
1. Rod-shaped - bacillus
2. Spheres - coccus, cocci
3. Comma-shaped cells - Vibrio
4. Spiral or helical cells - spirillum, spirilla
Basic structural components of the bacterial cell are the cell wall, the plasma membrane, nucleoid DNA and ribosomes. The cells may or may not have flagella, capsules, endospores, or specialized cell inclusion. The plasma membrane plays a dominant role in cellular physiology, as does DNA in cellular reproduction. Structurally the plasma membrane consists of a bilayer of lipids, with fatty acids attached to glycerol, commonly to glycerol phosphate. The fatty acids are hydrophobic and the glycerol moieties, hydrophilic. In water the hydrophobic ends line up in apposition to each other, and the glycerol ends, toward water. Proteins are embedded in this double layer. They provide the membrane with a wide variety of enzymes and cofactors, some of which are common to all organisms while others are unique to subgroups or species. The main difference between gram-positive and gram-negative cell walls is the presence of the peptidoglycan or murein layer. (Fig. 3) In gram positive (G+) bacteria, the plasma membrane is surrounded by a thick cell wall. Gram negative (G-) bacteria have a much thinner cell wall, which in turn is surrounded by an outer cell membrane that is very similar to the plasma membrane. In G+ bacteria, the cell wall contains peptidoglycan and teichoic acids. Cell wall of G- bacteria contains peptidoglycan but lack teichoic acid. In the four step gram stain, the alcohol step is believed to shrink the thick petidoglycan layer of G+ bacteria sufficiently to prevent washout of crystal violet. In G- bacteria, the thin peptidoglycan layer has initially larger pores and alcohol does not lock in the crystal violet. The destained cells can then accept the counterstain.
The bacterial endospore is the most resistant life form that we know of. Materials must be autoclaved in order to kill endospores. Autoclaving = steam under pressure. You need at least 121° C (254° F) at 15psi for 15 - 20 minutes.
Human health aspects of endospore production by bacteria:
Family I. Bacillaceae Genus I. Bacillus - aerobic, gram-positive, endospore-forming rods Bacillus anthracis – causative agent of anthrax
Endospores get their durability in part from a very thick spore coat rich in cross-linked dipicolinicacid:
The flagellum is the organelle of locomotion in the bacteria. It is a fine hollow tube of a helically wound protein called flagellin.
Flagella may be borne in several configuations:
Bacteria are usually the most abundant group in soils in terms of numbers. This does not imply that they comprise the greatest biomass. Because of their small size, it takes many more cells to equal a unit of biomass of a larger organism. Thus bacteria account for appreciably less than ½ of the total microbiological cell mass in a soil.
Winogradsky devised an ecological classification of the soil bacteria population. Soil Bacteria can be divided into two broad categories:
|Soil||Bacterial Mass (x)||Substrate flow (ds/dt)||Maintenance energy|
|Grassland||60 g m-2||0.006 g h-1 m-2||4.8 g h-1 m-2|
|Forest||500 kg *||0.9 kg h-1||0.9 kg h-1|
|Forest||7.3 kg ha-1||0.9 kg h-1 ha-1||0.584 kg h-1 ha-1|
|Forest||36.87 kg ha-1||0.806 kg h-1 ha-1||2.95kg h-1 ha-1|
|Silt Loan||8.4 kg ha-1||0.118 kg h-1 ha-1||0.672 kg h-1 ha-1|
* includes fungi
Bacterial growth is ideally exponential because the size of the population is related to an exponential function of the base 2.
b = (a) x 2n
log b = log a + n log 2
0.301n = log b - log a
n = log b - log a
g = t/n
g = 0.301t / log b - log a
t = selected time interval
g = generation time
Generation times of microbes vary quite widely ranging from as little as 9 minutes, to as long as several days.
Lag Phase - a period of adaptation to new surroundings (nutrients, environment, etc,.)
Log or exponential phase - population is growing logarithmically (2,4,8,16,32,64…)
Stationary phase - period where reproduction = death rate. Zero population growth: A reflection that the environment has become unfavorable, nutrients are depleted, toxins are accumulating, O2may be exhausted, etc.
Death phase - cells are dying faster than they are reproducing. Conditions have become unsustainable for continued growth and survival.
These properties are very difficult to determine in a system so highly diverse and complex as the soil.
Sampling errors are serious limitations:
When distribution may vary from one cm to the next; how does one adequately sample a large field or even a square meter for microbial counts? This is often the most difficult decision to make.
A reasonable method is to combine many replicate cores into a composite sample. It is better to use many subsamples than numerous replicate plates per dilution since the variation among duplicate samples is far greater than that among duplicate plates or dilutions.
A. Direct Microscopic Techniques
B.Determination of Numbers of Viable (Living) Bacteria
Direct microscopic counts usually give values 10 - 100x higher than viable counting techniques.
Plate counts of soil bacteria usually range from 105 - 2 x 108 cells per gram. Direct counts usually range from 108 - 1010 per gram. The number of bacteria frequently exceeds the combined numbers of all the other groups of soil microbes.
Estimates of Bacterial Biomass in Soils:
Assume: Each cell = 1 μm3 in volume. In a soil containing 108 bacteria per cm3 of space, the bacteria would occupy 108 bacteria x 1 μm3 per cell = 108 μm3 = 1 cm3 = 0.01% of total soil volume.
The average bacterial cell weights about 1.5 x 10-12 gram wet weight. Thus, using a direct microscopic count of 109 per cm3, 0.1% of the total soil volume would be bacterial protoplasm. If viable and microscopic counts are taken at 108 and 109 per gram, respectively, and the average cell weighs 1.5 x 10-12 gram, some 300 - 3000 kg live weight of bacteria is calculated to be present in each hectare of surface soil, which is, 0.015 - 0.05% of the total soil mass. Estimates by a number of methods indicate a range of 100 - 4000 kg / ha live weight of bacteria.
Counting Methods and Limitations
1. Plate Count - a preferred method for viable counts. Soil is diluted and aliquots of the dilutions are spead on agar plates or mixed into cooled molten agar (40°C). See Animation.
Look at Dilutions animation.
Limitations of plate counts:
Advantages of plate counts:
2. The Most Probable Number Technique
A tube dilution technique in which series of replicate dilution tubes are scored positively or negatively for a given trait, i.e. growth or no growth, gas or no gas, etc. Used primarily to enumerage microbes that don't grow well on solid culture media and thus are not amenable to plate counting.
A code number is derived and the most probable number of cells is determined from MPN statistical tables.
3. Direct Microscopic Counts
A known volume of a given soil dilution, usually 0.01 ml of a 1/10 dilution as in the Breed method is placed in a specialized counting chamber, and replicate squares of known area are scored for #'s of bacteria. By appropriate calculations, the # of cells can be determined. Petroff-Hausser Counting Chamber is an apparatus used for making direct counts.
Primary Environmental Variables:
Bacteria can be divided into 3 groups based on temperature requirements for growth.
Temperature also governs rate of biochemical reactions - Q10 values of biological systems are usually about 2, i.e., for every 10° C increase in termpaerature, the reaction/growth rate doubles - up to a a point.
Community size in mineral soils is directly proportional to the organic matter content. Organic amendments generally increase the soil population markedly. See curves below for bacterial numbers and oranic matter content vs. depth in soil.
Organisms often parallel the distribution of organic matter in the soils.
Most natural environments have a pH value between 5 and 9, and organisms with optima in this range are most common. Yeast and fungi grow best at slightly acid conditions. Blue-green algae (cyanobacteria) grow best under slightly alkaline conditions. Most bacteria have a relatively narrow pH range (3 or 4 units) although a few grow over wide ranges. Some species range from 2 - 8. Those that grow at very low pH values are usually obligate acidophiles, unable to grow at neutral pH and evern killed by such pH values. Example: Acidithiobacillus thiooxidans - a sulfur oxidizing chemoautotroph.At the other extremes are the alkalophiles that grow at very high pH values, 10 - 12.
Intracellular pH is almost always near neutrality. Chlorophyll, DNA, and ATP are acid labile. RNA and phospholipids are alkali labile. In the cell, pH must be carefully regulated. pH also exerts indirect effects due to the ionization of organic compounds in the medium or environment. Non-ionized forms of most compounds can penetrate the cells more readily than can the ionized forms. This can have an effect on uptake of nutrients.
Modification of pH by Organisms
The sources from which an organism derives its cell C and energy are useful for describing basic physiological differences among bacteria as well as among organisms generally. Those using light as their energy source are phototrophic; those deriving their energy from a chemical source chemotrophic. If CO2 is used as the cell C source, the organism is termed autotrophic. If cell C is derived principally from an organic substrate, the organism is heterotrophic. The combining terms litho and organo refer to the electorn donor as either inorganic or organic, respectively. The majorities of known bacterial species are chemorganictrophic and are commonly referred to as heterotrohs. Photolithotrophs include the higher plants, algae, cyanobacteria, and purple and green sulfur bacteria. Chemolithotrophs use diverse energy sources, e.g., NH4+, NO2- , Fe2+, S2-, and S2O3-. The obligate chemolithotrophs use the same basic physiological pathway (the Krebs cycle) found in most other organism in metabolizing their own cell constituents. Their apparent inability to use an external source of organic C is possibly linked to lack of permeases to move organic molecules across cell membranes. Organic molecules must be manufactured within the cell.
There are other terms for characterizing nutritional differences among bacteria. In the autochthonous and zymogenous separation first used by Winogradsky, autochthonous designated those organisms growing in soil containing no abundant supply of easily oxidizable substrate. Zymogenous organisms were those showing rapid growth when fresh residues were added to soil. This terminology is no longer widely used. Organisms that are capable of using (fixing) dinitrogen are termed diazotrophic.
Oligotrophy and copiotrophy are terms now in general use. Oligotrophic organisms are those growing in a nutritional environment of extremely low organic availability. Copiotrophic organisms require high levels of organic nutrients for growth. A favorable nutrient concentration range for oligotrophs is 1 to 15mg soluble C liter-1. Copiotrophs are routinely cultured at about 1000 mg soluble C liter-1.
Response is based on an integration of moisture, aeration, and organic matter effects on growth and numbers.
Plowing and tillage drastically alter the soil environment. Changes vary with the type of operation, soil depth and especially the type of crop residues that are turned under.
Effects are due to:
A variable composed of several well-defined primary variables including:
Cell numbers are usually greatest in the spring and autumn, and a decline occurs during the hot, dry summer months. Burst in spring is due to warming of the soil and availability of nutrients. In autumn the increase is due to more favorable moisture status, and the availability of residues, i.e. roots and above ground tissues.
Seasonal fluctuations in the bacterial population are closely related to changes in moisture and temperature. Root exudation that provides carbon sources for microbial growth correlates with growing season, etc.
In temperate regions, bacteria are almost entirely within the top meter and predominantly in the top few centimeters.
At the very surface the population is sparse due to low moisture and possible bactericidal action of sunrays. See graph under organic carbon above. In shaded forest or orchard soils the highest numbers are frequently in the surface 1-2 cm where litter and organic matter accumulate.
In organic soils the numbers of bacteria frequently fail to fall appreciably with depth sometimes being greater at 160 cm than at the surface. Fluctuations with depth are largely controlled by the availability of organic carbon and by aeration status.
This phylum contains the majority of the traditionally known G- bacteria. It is extremely diverse, embracing heterotrophs, chemolithotrophs, and chemophototrophs. Some genera are anaerobic; others are aerobic.
Many members of the subdivision are capable of forming intimate relationships with eukaryotes. Those closely associated with plant roots are called rhizobacteria; the genus Pseudomonas is best known. Pseudomonads are small, straight or slightly curved rods with one or several polar flagella. They degraded a wide variety of sugars, amino acids, alcohols, and aldoses. Some species can degrade higher molecular weight compounds such as humic acids and pesticides. Pseudomonads commonly produce diffusible fluorescent pigments containing siderophores that have great affinity for Iron. Some fluorescent species have been used for the biological control of soilborne phytophathogens. In alkaline soils of low Fe availability, the bacteria sequester Fe, depriving the pathogen of this nutrient. Other pseudomonads are destructive phytopathogens, forming wilt-inducing cellulases and pectinases. On the beneficial side, some produce growth stimulants, such as ethylene, and indoleacetic acid. Highly beneficial are the diazotrophic rhizobia that form nodules on the roots of legumes. These bacteria have been designated species of Rhizobium and Bradyrhizobium. They are classified as pseudomonads from a molecular basis.
The purple, sulfur-oxidizing bacteria contain carotenoids and bacteriochlorphylls in the cytoplasmic membrane and in intracytoplasmic membranes that are continuous with the cytoplasmic membrane. When growing on reduced sulfur compounds, on subgroup (Chromaceae) forms globules of elemental S inside the cell, then oxidizes the S to sulfate. The second sub-group (Ectothiorhodospiraceae) forms S globules outside the cell, then oxidizes the S to sulfate. Chromatium and Ectothiorhodospira are the best-known genera.
The purple nonsulfur bacteria are generally photoheterotrophic and anaerobic. Some are photolithotrophic and use molecular hydrogen. Others used sulfide or elemental S as the electron donor, with CO2 as the source of cell C. Most species, however, are intolerant to Sulfide even at low concentrations; they depend on sulfate for their cell S. The purple nonsulfur bacteria are widely distributed in waters and moist soils. Six genera are recognized; rhodospirillum, Rhodopila, Rhodobacter, Rhodopseudomonas, Rhodomicrobium, and Rhodocyclus. Also included in the purple bacteria and of great importance in ecosystem process are the nitrifying bacteria and the Fe and Mn oxidizers.
These bacteria are obligately photolithotrophic and anaerobic and capable of using sulfide or elemental S as the electron donor. In sulfide oxidation, S0 globules are formed outside the cell, then absorbed and further oxidized to sulfate. All genera photoassimilate simple organic substances in the presence of sulfide and bicarbonate. In culture, strains of the bacteria are either grass green or chocolate brown. The green species contain chlorphylls c and d and small amounts of a; the major carotenoid is chlorobactene. The browns contain bacteriochlorophyll e, a small amount of bacteriochlorophyl a and isorenieratane. The photosynthetic pigments are located in the cytoplasmic membrane and in chlorosomes inside of and attached to the membrane. The green sulfur bacteria occur in a variety of anaerobic and sulfide-containing muds and waters, both fresh and marine. The brown strains are found in the sulfide containing deeper layers of wetlands and ponds. In stratified lakes the green sulfur bacteria are usually found below the purple sulfur bacteria.
The two traditionally known genera of sporogenic bacilli are Bacillus (Figure 1)and Clostridium (Figure 2) . The former is aerobic and catalase positive the latter is anaerobic or microaerophilic and catalsase negative. The bacterial endospore is a dormancy structure formed within a mother cell and capable of remaining viable long after the death of the mother cell. Most endospores can survive heating to 70°C, some to even higher. In the genus Bacillus, several species produce lytic enzymes and antibiotic, notably polymyxin, gramicidin and bacitracin.
A crystalline toxin of Bacillus thruingiensis, known in the marketplace as Bt, is widely used for the biological control of insects. Clostridium butyricum and C. pasteurianum are nonsymbiotic diazotrophs. C. tetani, C. botulinium, and B. anthracis are well-known pathogens.
The cyanobacteria are obligate phototrophs with an oxygenic photosynthesis similar to that of higher plants. There were formerly classified as algae, being designated as the blue-green algae. The cyanobaceria occure in unicellular, colonial, filamentous hereocystous and filamentous branching forms. Cell diameters usually fall within the range of 1.0 and 10µm, but occasionally are as small as 0.5 or as large as 50µm. The cellular cytoplasm is structured with paired photosynthetic lamellae termed thylakoids. Their membranes bear the photosynthetic granules.
Cyanobacteria have a ubiquitous distribution in terrestrial, freshwater, and marine habitats (Figure 3). They are light dependent, but some can adapt to low levels of light intensity. Cyanobacteria are found in hot springs at 74°C and in polar deserts. Free sulfide is tolerated and sometimes used. On soil parent materials such as bare rocks and sand, cyanobacteria are often the primary colonizers, either alone or as symbionts of fungi in lichens. They also occur as symbionts or associates of algae, water ferns, and angiosperms.
Archaea are separable from bacteria both by their molecular phylogeny and by their phenology. Taxonomically the Archaea are divided into two kingdoms and five subgroups. In general the archaea are tolerant of extremely harsh environments. Cell membranes of archaea are unique. Their branch-chained, ether-linked lipids differ greatly from those of all other life forms. The basic structure is a 5-C isoprene unit. These are linked to form chains of up to 20 C sometimes as many as 40 C chain. Chains are ether linked to glycerol, not ster linked as in bacteria and eucarya. Halophiles have glycerol diether units; methanogens have mixed glycerol- diether and diglycerol-tetraether units. In thermophilic archaea, the tetraether membrane is predominant.
The halophiles require high concentrations of NaCl. Most grow best at 3-4 M. To counterbalance the external NaCl, cells accumulate a high internal concentration of KCl. Many species of halophiles produce a red carotenoid pigment that gives them protection from strong sunlight; it also makes them responsible for red discoloration on salted fish and in saline waters.
Halophiles are aerobic chemorganotrophs and most are culturable only in nutrient-rich media, with peptides and amino acids as the C source. Many halophiles use light to drive cellular metabolism. In photometabolism, cells use the pigment retinal; they lack the plant and bacterial chlorophylls. Bacteriorhodospsin, the retinal-containing protein, moves protons from the cell interior to the outside thus creating a membrane potential for use as a source of cell energy.
The strictly anaerobic methanogens are unique in their ability to produce CH4 as a metabolic product. Methane emissions occur from swamps, marshes, and marine sediments; from the intestinal tracts and rumens of animals; and from sludge digesters in sewage plants. In sewage digesters methane is produced in such quantity that it is commonly harvested for commercial use. Emissions from natural sources escape to the atmosphere. Methanogens do not use sugars as a source of cell C. Carbon dioxide is commonly the C source. The C atom of CO2sub>is reduced to CH4 by electrons derived from hydrogen.
Four genera included as extreme thremophiles are Archaeoglobus, Thermoplasma, Thermococcus, and Pyrococcus. Archaeoglobus is strictly anaerobic and chemorganotrophic, catabolizing sugars and simple peptides, using sulfate as the electron acceptor, reducing it to sulfide.
Thermoplasma is facultatively anaerobic and grows best at pH 1.5 and 60°C. Aerobically it grows poorly on sugars. The genus does not have a cell wall external to the cell membrane.
Thermococcus and Pyrococcus are very similar except for a difference in their growth temperatures. Thermococcus grows optimally at 83°C and Pyrococcus at 100°C. Both are obligate anaerobes and chemorganotrophs, using sugars and complex carbon compounds and reducing S0 to H2S. Cells can initiate growth in the absence of S0, but H2 accumulates and inhibits further growth.