Phospholipid Fatty Acid (PLFA) Science, Inovation, Networks

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2 Phospholipid Fatty Acid (PLFA)

3 Specific components of cell membranes that are only found in intact (viable) cells and rapidly degraded after cell death (White et.al, 1997 in Yao et.al, 2000) Cell membrane

4 Catalyst Triacylglycerol (Triglyceride) Methanol Glycerol Fatty acid methyl esters (FAME)

5 Microbial Groups Gram (+) Bacteria (i15:0, a15:0, i16:0, i17:0, a17:0, 15:0, 17:0) ) Gram (-) Bacteria (16:1ω7, cy17:0, 18:1ω7, cy19:0) Fungi (18:2ω6,9 & 18:1ω9) Actinomycetes (10Me17:0; 10Me18:0; 10Me19:0) Protozoa (20:2ω6,9c; 20:3ω6,9,12c; 20:4ω6,9,12,15c) ) Arbuscular Mycorrhizal Fungi (C16:1ω5) Stress Condition Ratio of Specific PLFAs 16:1ω7t/16:1ω7 cy17:0/16:1ω7 cy19:0/18:1ω7

6 PLFA Extraction Day 1 Extract lipid from soil Day 2 Separated soil lipid extract into 3 classes with SPE cartridge (SiOH) Day 3 Transesterification

7 PLFA Extraction (Day 1) 2 g soil Add 3.6 ml Buffer fosfat, 4 ml CHCl 3, 8 ml Methanol Shake for 1 h Centrifuge at Decant supernatant into 2500rpm for 10 min separatory funnel that contain CHCl3 and Buffer fosfat Leave to stand overnight B

8 Drain organic phase into centrifuge tubes PLFA Extraction (Day 2) Dry extract lipid with N 2 gas Redissolve lipid with CHCl 3 Separated lipid into 3 classes with SPE cartridge (SiOH column) CHCl3 Neutral lipid Acetone Glycolipidli id Methanol Phospholipid Dryphospholipid with N 2 gas B

9 PLFA Extraction (Day 3) Dried PLFA Add 1 ml MeOH Toluene and 1 ml Methanolic KOH Measured with Gas Chromatography Incubate for 15 min at 37 C Redissolve e FAME with Istd C19:0 Add 2 ml Hexane CHCl 3, 1 ml acetic acid 1M and 2 ml DW Dry extract FAME with N 2 gas B Centrifuge at 2000rpm for 5 min Transfer upper phase (FAME extract) to glass tube

10 Number of publications found when searching ISI web of science for PLFA AND soil There was one in 1991 and three in Totally there were 708 hits.

11 Changes in the PLFA profile represent changes in the total soil microbial community, and it has been used to compare different land use systems and crop management (Bossio et.al, 1998 in Yao et.al, 2000). Microbial diversity (community structure) has also been recommended as a biological indicator of soil quality. The stress indicator ratio of cy19:0 to 18:1ω7 increased throughout the experiment in Grassland and Vegetable soil in response to stimulated tillage (Calderon et.al. 2000)

12 The use of PLFA analysis is an efficient way to rapidly screen whether the fungal or bacterial part of the soil community has been affected by a treatment. The relative abundance of fungi and bacteria, often referred as the fungal/bacterial ratio, can be calculated from the amounts of PLFAs specific to these respective groups (Frostegård and Bååth, 1996) and has been used widely for comparing soils and treatments.

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15 MBC (Wang et al., 2011) Represented the living part of the organic matter in soils Indicative of the size and diversity of the soil microbial community Methods Fumigation Incubation (Jenkinson and Powlson, 1976) Fumigation Extraction (Vance et al., 1987) Substrate Induced Respiration (Anderson and Domsch, 1978)

16 Microbial Biomass Carbon (Fumigation-Extraction Method) Soil Fumigate soil under vapour CHCl 3 -free ethanol for 24 h Extract soil with K 2 SO 4 0,5 M Measured with TOC Analyzer

17 Dehydrogenase Activity Dehydrogenase activity as a general measure of viable microorganisms is often closely related to microbial populations in soils. Dehydrogenase activity of the soil is considered to be an indicator of the microbial redox system and of the oxidative activities of the soil (Trevors, 1984) Dehydrogenase activity has also been employed in soils affected by heavy metals and pesticides, as well as for the diagnosis of the degree of recovery of degraded soils.

18 Dehydrogenase Activity 5 g moist soil + 2 ml TTC + 2 ml tris buffer Incubated at 37 C for 24 h Extract with methanol Read absorbans at λ=485 nm

19 β Glucosidase The rate limiting enzyme in the microbial degradation of cellulose to glucose, plays a crucial role in the C cycle of soils (Perez de Mora et al., 2005) Sensitive to soil management effects (Bandick and Dick, 1999) Has been suggested to be an integrative measure of physicochemical and biological soil properties (Turner et al., 2002).

20 β Glucosidase 1 g moist soil + 4 ml MUB ph ml PNG Incubated at 37 C for 1 h Add 1 ml CaCl ml Buffer Tris ph 12 Shake and filtered with Whatman filter paper No. 5 Read absorbans at λ=400 nm

21 β Glucosaminidase Β Glucosaminidase is one of the enzymes that play a major role in N mineralization in soils Degrade chitin (one of the most abundant biopolymers on earth serving as an important transient pool of organic C and N in soils)

22 β Glucosaminidase 1 g moist soil + 4 ml Acetate Buffer ph 5,5 + 1 ml PNNAG Incubated at 37 C for 1 h Add 1 ml CaCl ml Tris Buffer ph 12 Shake and filtered with Whatman filter paper No. 5 Read absorbans at λ=405 nm

23 Turner et.al., 2002 Substrate induced β Glucosidase activity was positively correlated with concentrations of clay, total carbon and microbial carbon. This suggests that substrate induced β glucosidase activity is an integrative measure of physico chemical and biological i l soil properties and may have applications in monitoring biological soil quality.

24 Gajda & Martyniuk, 2005 The activities of dhd dehydrogenase and phosphatase h enzyme and microbial biomass C and N content in the monoculture soil were generally significantly lowerthanthoseinthesoilfromtheorganicand conventional short rotation systems, indicating that substantial disturbances may occur in the microbial activity of the monoculture soil

25 Allison et.al., 2007 Enzyme efficiencies are still negatively correlated with nutrient availability In mineral soil, efficiency of enzymes was negatively correlated with soil nutrient availability In organic soil, efficiency of C,N and S hydrolyzing y enzymes was positively correlated with soil P, and not negatively correlated with availability of C, N and S, whereas efficiency of P hydrolyzing hd enzymes was negatively correlated with soil P

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27 Nematode Multicellular animals Colorless, unsegmented Free living, predaceous, or parasitic Feed on bacteria, fungi, and other nematodes Vary in sensitivity to pollutants and environmental disturbance Represent a central position in the soil food web and correlate with ecological processes such as nitrogen cycling, decomposition and plant growth can used as bioindicators of soil health or condition

28 Nematode Extraction (Cobb s Method) 100 g soil + 1 L water Collecting pan

29 Nematode Extraction (Cobb s Method) Collecting pan Add 85 ml water Let stand for hours Nematode suspension poured into a 100 ml beaker Let stand suspension for 2 hour Filter Observed under microscope Remove some water till remaining ±20 ml

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