Objectives:

1. Chapter 4 - The Terrestrial Environment • Objectives: • Understand the terrestrial environmental from an integrated physical, chemical and biological perspective. • Define a surface soil, the vadose zone, and the saturated zone. • Define components of soil discussed in class such as texture, pore size distribution, organic matter, soil structure, interaggregate and intraaggregate pores, cation exchange, soil water potential. • Understand how soil water potential relates to microbial activity. • Understand the basics of contaminant sorption and microbial sorption. • Understand how microbial activity can influence the soil atmosphere. • Be able to describe the types, numbers, and relative activities of microbes found in surface soil, vadose zone, and saturated zone environments. • Discuss the respective competitiveness of the bacteria, • actinomycetes, and fungi in soil.

2. Surface soils Vadose zone Saturated zone shallow aquifers intermediate aquifers deep aquifers

3. Components of a typical soil 1) 45% mineral (Si, Fe, Al, Ca, K, Mg, Na) The two most abundant elements in the earth’s crust are Si (47%) and O (27%) Quartz = SiO2 Clay minerals are aluminum silicates Nonsilicates = NaCl, CaSO4 (gypsum), CaCO3 (calcite) OM 2) 50% pore space 3) 1 to 5% organic matter Mineral Pore space

4. Soil texture – this defines the mineral particle sizes that make up a particular soil. particle diameter Surface to volume ratio range (mm)(cm2/g) Sand: 0.05 – 2 mm 50 Silt: 0.002 – 0.05 mm 450 Clay: 0.0002 – 0.002 mm 10,000

5. Texture and pore size distribution The amount of clay and organic matter in a soil influence the reactivity of that soil because they both add surface area and charge. Because large amounts of clay make the texture of the soil much finer, the average pore size is smaller. Similarly fluids like water move more easily through large pores, not because the water molecules are too large, but because there is less resistance to water movement through larger spaces. Pore size distribution is important when one considers movement of fluids and of microbes through a porous medium. Protozoa and bacteria will have difficulty moving through even sandy porous media.

6. Pore size 5% of the mean pore diameter 20 um 0.6-20 um 0.02–0.6 um Filtration is important when the size of the bacterium is greater than 5% of the mean diameter of the soil particles

7. Organic Matter The major input of organic matter in soil is from plant, animal, and microbial biomass. Humus is the ultimate product of degradation of organic matter. Humus is aromatic in character. This is because the humus backbone is derived from the heterogeneous plant polymer lignin which is less readily degradable than other plant polymers (cellulose and hemicellulose). Core molecules for organic humus Humus has a three dimensional sponge-like structure that can absorb water and solutes in the water. Humus is only slowly utilized by soil organisms and has a turnover rate of 1 to 2% per year. In general soils with higher organic matter contents have higher numbers of microbes and higher levels of activity.

8. Humus shares two properties with clay: it is highly charged and it has a large surface area to volume ratio. The quantity of organic matter found in soil depends on climate. Soils found in temperate climates with high rainfall have increased levels of organic matter. Levels of organic matter found in soil range from essential no organic matter (Yuma, AZ) to 0.1% organic matter (Tucson, AZ) to 3 to 5% organic matter (midwest) to 20% organic matter (bogs and wetlands). Bogs and wetlands Organic matter > 20% Bogs cover 5 – 8% of the terrestrial surface Why do peat bogs have very low microbial activity? (see Info Box 4.2)

9. Cross-section Surface Soils 10 structure = soil particles + organic matter (humus) + roots + microorganisms 20 structure = aggregate or ped = stability

10. Soil aggregates are formed and stabilized by clay-organic complexes, microbial polysaccharides, fungal hyphae and plant roots. See Info Box 4.4 for a special case of aggregation, cryptobiotic crusts.

11. Soil aggregates are associated with relatively large inter-aggregate pore spaces that range from um to mm in diameter. Each aggregate also has intra-aggregate pore spaces that are very small, ranging from nm to um in diameter. Intra-aggregate pores can exclude bacteria (called micropore exclusion). However, after a spill, contaminants can slowly diffuse into these pores. This creates a long-term sink of pollution as the contaminants will slowly diffuse out again.

12. Just how many pores are there? Assume a soil aggregate that is 2 x 2 x 2 mm. Further assume that the volume of the aggregate is 50% pore space. How many pores of diameter 15 um does the aggregate have? How many pores of 50 um? (the volume of a sphere is: 4/3π r3) 2 mm 2 mm 2 mm Calculation for 15 um pores: The volume of the aggregate is 2 mm x 2 mm x 2 mm = 8 mm3 Pore space is 50% of 8 mm3 = 4 mm3 A pore of 15 um diameter has volume = 4/3 π (7.5 um)3 = 1.77 x 103 um3 4 mm3(1000 um)3 / 1.77 x 103 um3 = 2.3 x 10 6 pores of 15 um per aggregate! mm3 pore

13. Where are the bacteria? In soil 80 to 90% of the bacteria are attached to surfaces and only 10-20% are planktonic. Cells have a patchy distribution over the solid surfaces, growing in microcolonies. Colony growth allows sharing of nutrients and helps protect against dessication and predation or grazing by protozoa.

14. Interaction of contaminants and microbes with soil surfaces Soils have an overall net negative charge that comes from clay oxides, oxyhydroxides, and hydroxides. The negative charge attracts positively charged solutes from the soil solution in a process called cation exchange. Organic matter also provides a net negative charge and adds to the cation exchange capacity of a soil. Normally, soil cations such as Na+, K+, or Mg2+ bind to cation exchange sites. However, when a positively charged metal contaminant such as lead (Pb2+) or an organic contaminant are present they can displace these cations. This leads to sorption of the contaminant by the soil.

15. Cation Exchange

16. Similarly, bacteria are sorbed to soil. In this case the bacterium, which like the soil has a net negative charge, is sorbed through a cation bridge.

17. A second mechanism for sorption of contaminants is hydrophobic binding. Hydrophobic sites on the soil surface are created when organic matter is present. Polar groups in the sponge-like organic matter structure face the outside while non-polar groups are in the interior of the sponge. Nonpolar molecules are attracted to the nonpolar sites in the organic matter resulting in hydrophobic binding.

18. The soil solution is a constantly changing matrix composed of both organic and inorganic solutes in aqueous solution. Soil Solution

19. Water movement and soil water potential Soil water potential depends on how tightly water is held to a soil surface. This in turn depends on how much water is present. Surface forces have water potentials ranging from –10,000 to –31 atm. Capillary forces have water potentials ranging from –31 to –0.1 atm. Optimal microbial activity occurs at approximately -0.1 atm. At greater distances there is little force holding water to the surface. This is considered free water and moves downward due to the force of gravity.

20. Composition (% volume basis) Location Nitrogen (N2) Oxygen (O2) Carbon Dioxide (CO2) Atmosphere Well-aerated surface soil Fine clay/saturated soil 78.1 78.1 >79 20.9 18 - 20.5 0 - 10 0.03 0.3 – 3 Up to 10 Soil atmosphere The composition of the earth’s atmosphere is approximately 79% nitrogen, 21% oxygen, and 0.03% carbon dioxide. Microbial activity in the soil can change the local concentration of these gases especially in saturated areas.

21. Microorganisms in soil – an overview • minor role as primary producers • major role in cycling of nutrients • role in soil formation • role in pollution abatement

22. Highest numbers Highest biomass Numbers and types of microbes in typical surface soils Bacteria Culturable counts 106 – 108 CFU/g soil Direct counts 107 – 1010 cells/g soil Estimated to be up to 10,000 species of bacteria/g soil Actinomycetes Culturable counts 106 – 107 CFU/g soil Gram Positive with high G+C content Produce geosmin (earthy smell) and antibiotics Fungi Culturable counts 105 – 106/g soil Obligate aerobes Produce extensive mycelia (filaments) that can cover large areas. Mycorrhizae are associated with plant roots. White rot fungus, Phanerochaete chrysosporium is known for its ability to degrade contaminants.

23. Comparison of bacteria, actinomycetes, and fungi BacteriaActinomycetes Fungi Numbers highest intermediatelowest Biomass --- similar biomass --- largest Cell wall --- PEP, teichoic acid, LPS --- chitin/cellulose Competitiveness mostleast intermediate for simple organics Fix N2YesYesNo Aerobic/Anaerobic bothmostly aerobicaerobic Moisture stress least tolerantintermediatemost tolerant Optimum pH 6-86-86-7 Competitive pH 6-8>8<5 Competitiveness all soilsdominate dry,dominate high pH soilslow pH soils

24. ? ? ? Surface soil Vadose zone Shallow saturated zone Bacterial numbers and activity in surface soil, the vadose zone, and the saturated zone Example 1: A shallow core Konopka and Turco (1991) compared microbial numbers and activity in a 25 m core that included surface soil, vadose zone, and shallow saturated zone samples. Site was a 40 year old corn field at Purdue University

25. Culturable counts (10-3 CFU/g) AODC (10-7 cells/g) Phospholipid (ug/g) Compare the microbial numbers in the surface, vadose zone, and saturated regions.

26. Surface soil sample glucose phenol 80 60 40 20 0 0 8 16 24 32 Vadose zone sample 80 60 40 20 0 14CO2 evolved as a % of the carbon added 0 8 16 24 32 80 60 40 20 0 Saturated zone sample 0 8 16 24 32 Days Compare the microbial activity in the three regions in terms of: 1) lag time 2) growth rate 3) cell yield.

27. Example 2: The deep vadose zone A 70 m core was taken in the Snake River Plain in Idaho (Colwell, 1989). Compare the direct and culturable counts between the surface samples and the deep vadose zone samples. TABLE 4.11 A comparison of microbial counts in surface and 70-m unsaturated subsurface environments aCFU, colony-forming units.

28. Example 3: The deep saturated zone In 1987, a 470 m core was taken in the southeast coastal plain in South Carolina (Fredrickson et al., 1991). Culturable counts ranged from 103 to 106 CFU/g in a permeable sandy sample retrieved from between 350 and 413 m. Culturable counts were lower (non-detect to 104 CFU/g) in a low permeability sample taken between 450 and 470 m. More recently, (2001-2006), a series of water samples were taken from the saturated zone at depths of 0.72 - 3 km in the Witwatersrand Basin in central South Africa ( Gihring et al ., 2006 ). Total microbial numbers in the samples were estimated to be as low as 103 cells/ml. Diversity was low as shown by analysis of the 16S rRNA gene, which generated only an average of 11 bacterial OTUs from all the samples. Compare this to surface soils that have up to 6300 OTUs! Compare the microbial counts measured in surface, vadose zone, and saturated zone samples presented in the 3 examples. What do these counts imply for activity in each of these regions? What do these counts imply for diversity in each of these regions?

29. Summary and Reality Check Despite the fact that there are microbes present in most subsurface samples, often in high numbers, the level of microbial activity in the deep subsurface is very very low when compared to activity in surface soils or in lake sediments.