Science, Inovation, Networks · . 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

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Phospholipid Fatty Acid(PLFA)


Specific components of cell membranes that are onlyfound in intact (viable) cells and rapidly degraded aftercell death (White et.al, 1997 in Yao et.al, 2000)

Cell membrane


Catalyst

MethanolMethanolTriacylglycerolTriacylglycerol Fatty acid methyl estersFatty acid methyl estersGlycerolGlycerolMethanolMethanolTriacylglycerol(Triglyceride)Triacylglycerol(Triglyceride)

Fatty acid methyl esters(FAME)

Fatty acid methyl esters(FAME)

GlycerolGlycerol


Stress ConditionMicrobial Groups

Ratio of Specific PLFAs16:1ω7t/16:1ω7

Gram (+) Bacteria (i15:0, a15:0, i16:0, i17:0, a17:0, 15:0, 17:0)

cy17:0/16:1ω7cy19:0/18:1ω7

)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 4 6 9 12 15 )20:4ω6,9,12,15c)Arbuscular Mycorrhizal Fungi (C16:1ω5)


PLFA Extraction

• Extract lipid from soilDay 1

• Separated soil lipid extract into 3 classes with SPE cartridge (SiOH)Day 2

• TransesterificationDay 3


PLFA Extraction (Day 1)

2 g soil

Add 3.6 ml Buffer‐fosfat, 4 ml CHCl3, 8 ml Methanol

Shake for 1 h

Centrifuge at 2500rpm for 10 min

Decant supernatant into separatory funnel that contain Leave to stand 2500rpm for 10 min p y

CHCl3 and Buffer‐fosfat overnight

B


B

Drain organic PLFA Extraction

( 2)g

phase into centrifuge tubes

(Day 2)

Dry extract lipid with N2 gas

Redissolve lipid Dry phospholipidSeparated lipid into 3

Redissolve lipid with CHCl3

Dry phospholipid with N2 gas

classes with SPE cartridge (SiOH column)

CHCl3 Neutral lipidA t Gl li id

B

Acetone GlycolipidMethanol Phospholipid


B

PLFA Extraction (Day 3)

Add 1 ml MeOH‐Toluene and 1 ml Methanolic KOH

Dried PLFA Measured with Gas

Incubate for 15 min at 37°C Redissol e FAME

Chromatography

Incubate for 15 min at 37°C Redissolve FAME with Istd C19:0

Dry extract FAME with N2 gas

Add 2 ml Hexane‐CHCl3, 1 ml acetic acid 1M and 2 ml DW

Centrifuge at 2000rpm for 5 min

B

Transfer upper phase (FAME extract) to glass tube


B

Number of publications found when searching ISI web‐of‐science for “PLFAAND soil” 2010‐11‐16. There was one in 1991 and three in 1993. Totally there were708 hits.


• Changes in the PLFA profile represent changes in the total soili bi l i d i h b d diffmicrobial community, and it has been used to compare different

land use systems and crop management (Bossio et.al, 1998 in Yaoet.al, 2000). Microbial diversity (community structure) has alsobeen recommended as a biological indicator of soil quality.

• The stress‐indicator ratio of cy19:0 to 18:1ω7 increasedThe stress indicator ratio of cy19:0 to 18:1ω7 increasedthroughout the experiment in Grassland and Vegetable soil inresponse to stimulated tillage (Calderon et.al. 2000)


The use of PLFA analysis is an efficient way to rapidly screenwhether the fungal or bacterial part of the soil communityhas been affected by a treatment The relative abundance ofhas been affected by a treatment. The relative abundance offungi and bacteria, often referred as the fungal/bacterialratio, can be calculated from the amounts of PLFAs specific toth ti (F t å d d Bååth 1996) dthese respective groups (Frostegård and Bååth, 1996) andhas been used widely for comparing soils and treatments.


MBC (Wang et al., 2011)

• Represented the living

Methods

• Fumigation‐Incubation part of the organic matter in soils

• Indicative of the size and

(Jenkinson and Powlson, 1976)

• Fumigation‐Extractiondiversity of the soil microbial community

(Vance et al., 1987)• Substrate‐Induced Respiration (Anderson and Domsch, 1978)


Microbial Biomass Carbon(Fumigation-Extraction Method)(Fumigation Extraction Method)

Fumigate soil under vapour CHCl3-free ethanol

for 24 hSoil

Extract soil with K2SO4 0,5 M

for 24 h

Measured with TOC

Extract soil with K2SO4 0,5 M

Analyzer


Dehydrogenase ActivityDehydrogenase Activity

• Dehydrogenase activity as a general measure of viablemicroorganisms is often closely related to microbialpopulations in soilspopulations in soils.

• Dehydrogenase activity of the soil is considered to be anindicator of the microbial redox system and of theoxidative activities of the soil (Trevors, 1984)

• Dehydrogenase activity has also been employed in soilsaffected by heavy metals and pesticides as well as for theaffected by heavy metals and pesticides, as well as for thediagnosis of the degree of recovery of degraded soils.


Dehydrogenase ActivityDehydrogenase 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


β‐Glucosidaseβ Glucosidase

• The rate‐limiting enzyme in the microbial degradation ofcellulose 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)b d b f• Has been suggested to be an integrative measure of

physicochemical and biological soil properties (Turner et al.,2002).)


β‐Glucosidaseβ Glucosidase

1 g moist soil + 4 ml MUB pH 6 + 1 ml PNGg p


Add 1 ml CaCl2 + 4 ml Buffer‐Tris pH 12

Shake and filtered with Whatman filter paper No. 5



β‐Glucosaminidaseβ 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 h l f dearth serving as an important transient pool of organic C and

N in soils)


β‐Glucosaminidaseβ Glucosaminidase

1 g moist soil + 4 ml Acetate‐Buffer pH 5,5 + 1 ml PNNAG


Add 1 ml CaCl2 + 4 ml Tris‐Buffer pH 12

Shake and filtered with Whatman filter paper No. 5



Turner et.al., 2002Turner et.al., 2002

• Substrate induced β‐Glucosidase activity wasSubstrate induced β Glucosidase activity waspositively correlated with concentrations of clay,total carbon and microbial carbon. This suggests thatsubstrate‐induced β‐glucosidase activity is anintegrative measure of physico‐chemical andbi l i l il ti d h li tibiological soil properties and may have applicationsin monitoring biological soil quality.


Gajda & Martyniuk, 2005Gajda & Martyniuk, 2005

h f d h d d h h• The activities of dehydrogenase and phosphataseenzyme and microbial biomass C and N content inthe monoculture soil were generally significantlythe monoculture soil were generally significantlylower than those in the soil from the organic andconventional‐short rotation systems, indicating thaty , gsubstantial disturbances may occur in the microbialactivity of the monoculture soil


Allison et.al., 2007Allison et.al., 2007

• Enzyme efficiencies are still negatively correlatedEnzyme efficiencies are still negatively correlatedwith nutrient availability

• In mineral soil, efficiency of enzymes was negatively , y y g ycorrelated with soil nutrient availability

• In organic soil, efficiency of C‐, N‐ and S‐hydrolyzing g y y y genzymes was positively correlated with soil P, and not negatively correlated with availability of C, N and

h ff f h d lS, whereas efficiency of P‐hydrolyzing enzymes was negatively correlated with soil P


NematodeMulticellular animals

Colorless, unsegmented

Free living predaceous or parasiticFree‐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 bioindicatorsof soil health or conditioncan used as bioindicatorsof soil health or condition


Nematode Extraction (Cobb’s Method)100 g soil + 1 L

water

Collecting pan


Nematode Extraction (Cobb’s Method)

Nematode suspension poured into a 100 ml

Collecting panpoured into a 100 ml

beakerLet stand for 16‐48 hours

Add 85 ml water

Let stand suspension for ≥2 hour

Filter

Remove some water till remaining ± 20 ml

Filter

Observed undermicroscope


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Science, Inovation, Networks · . 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