activity of exoenzymes in treated wastewater irrigated soils · jüschke & marschner activity...

Jüschke & Marschner

Activity of Exoenzymes in Treated Wastewater Irrigated Soils

Elisabeth JÜSCHKEMax-Planck-Institute for Biogeochemistry Jena, Germany

Bernd MARSCHNERInstitute of GeographySoil Science / Soil Ecology Bochum, Germany

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IntroductionIntroduction

Municipal37%

Agriculture56%

Industrial 7%

Israeli Ministry of Environmental Protection (2002)

Water use in Israel

Surfacewater and springs

23%

Treatedwastewater

(TWW) 21%

Groundwater56%

Water Commision (2000)

2010 already~ 50%

MinEnvir 2010


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2002 2003 2004 2005 2006 2007 2008 2009 2010

Trea

ted

was

tew

ater

[MC

M]

0

100

200

300

400

500

600

modified after Tal (2006)MCM = million cubic meter

Established and projected treated wastewater (effluent) usein Israel


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Treated wastewater is a source of organic carbon.

TWW – Treated WasteWaterFW - FreshWater

Water quality

* BOD 10 mg/L** COD 100 mg/L*

**

Upgraded Effluent QualityStandards (Jan 2010)

Parameter Unit TWW FW

EC dS/m 2.3 1.0

Cl mg/L 364 201

Na meq/L 21.4 4.3

Ca + Mg meq/L 6.1 4.1

pH 8.3 7.4

DOC mg/L 23.5 1.1

TOC mg/L 47.6 n.d.

BOD mg/L 59.9 n.d.

COD mg/L 234.0 n.d.

< 1

< 20

< 10

(Israel Ministry of Environment 2010)


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Materials and Materials and MethodsMethods

YagurMizra

Wadi Fara

HaMa´pil

Ramat HakoveshBazra

Arad

Acco

sampling sitesSoil Organic Carbon - profiles

Gaza

Sampling siteLand use FAO

classificationSand (%)

Clay (%)

SOC (%)

Ramat Hakovesh orchard Chromic Luvisol 89 6 0.9

HaMa´pil orchard Chromic Luvisol 82 11 1.8

Bazra orchard Chromic Luvisol 82 12 0.5

Acco orchard Vertisol 22 52 1.0

Wadi Fara field Vertisol 11 66 2.3

Gaza field Chromic Luvisol 86 7 0.1

Enzyme activity studies


grapefruit orchard

avocado orchard

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ResultsResults

SOC [%]

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5

Soil

dept

h [c

m]

-120

-100

-80

-60

-40

-20

0

FW soilTWW soil

Soil organic carbon content (SOC)

• first ~50 cm similar SOC-content

• less SOC in the subsoil (deeper ~50 cm)

Sampling site: Ramat Hakovesh


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modified after Kuzyakov et al. (2000)(a) acceleration of SOM decomposition – positive priming effect(b) retardation of SOM decomposition – negative priming effect

soil + substrate

12CO2from additional respiration

14CO2from substrate

12CO2from soilbasal respiration

(a)


14CO2from substrate

Suppressedbasal respiration

soil + substrate

(b)

soil


Min

eral

isat

ion


Theory of priming effects


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Incubation of soil samples with additions ofeasily available organic substrates

CO2 determination

MultiplexerKonduktometer

Computer

Respicond

0000,00 mS

1 96

soil sample

0.6 M KOH

14CO2

12CO2

addition of 14C-fructose or14C-alanine

conductometer

25°C

1 mL KOH for scintillation counting

Gosda, W. modified after Nordgren (1988)



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Treated wastewater is a source of organic carbon.

Effect on soil microbial communities and activities

enzyme-substrate-complex

productssubstrate

enzyme

Hypothesis: increased release of exoenzymes into the soiland alteration in mobilisation of nutrients

? Effects along soil profiles

organic carbon = substrate for soil microorganisms


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Measurements with the help of fluorescence substrates(4-MUF = 4-Methylumbelliferon and AMC = 7-amino-4-methylcoumarin)

4-MUF-α-D-Glycoside

α-D-Glycosidase degradation of soluble sugars and starch

A range of hydrolytic enzymes, involved in C, N and P cycles, wereinvestigated using a fluorimetric microplate assay (after Marx et al. 2001).

Acid phosphatase

Leucinaminopeptidase

Tyrosinaminopeptidase

Argininaminopeptidase

N-acetyl-ß-glucosaminidase

ß –Cellobiohydrolase

ß- Glucosidase

α –Glucosidase ß- Xylosidaseß- Glucuronidase


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ResultsResults

(2) - only two of three sampling sites showed significant differences between control and treated sample, therefore only these values were calculated

Soil depth Cmic Cmic/Corg q CO2 acc. CO2 SOCmin SOCmin SOCmin PE PE[cm] alanine fructose alanine fructose

0-10 + + - ++ ++ - ++ - ++ - + - -

10-20 ++ + - +++ +++ + ++ - - - - - -

20-30 + + + + - ++ + - -

30-50 + - - +++ + - - - - - (2) - (2)

50-70 + - - + - + - + +

70-100 ++ - - - + - - + - - - - - (2) - (2)

Bazra, Acco and Wadi Farano difference between TWW and FW

- - - TWW < FW (three sampling sites)- - TWW < FW (two sampling sites)- TWW < FW (one sampling site)

+ - - TWW < FW (two sampling sites) and TWW > FW (one sampling site)+ - TWW < FW (one sampling site) and TWW > FW (one sampling site)

++ - TWW < FW (one sampling site) and TWW > FW (two sampling sites)+ TWW > FW (one sampling site)

++ TWW > FW (two sampling sites)+++ TWW > FW (three sampling sites)


Student t-Test p<0.05

Soil microbiological parameter along 3 irrigated soil profiles- Bazra, Acco and Wadi Fara -


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ResultsResults



0-10 + + - ++ ++ - ++ - ++ - + - -

10-20 ++ + - +++ +++ + ++ - - - - - -

20-30 + + + + - ++ + - -

30-50 + - - +++ + - - - - - (2) - (2)

50-70 + - - + - + - + +

70-100 ++ - - - + - - + - - - - - (2) - (2)









Soil depth α-glu ß-Xyl N-acet ß-glucoro ß-cello ß-glu pho Leu Tyr Arg[cm]

0-10 + ++ + ++ ++ + - ++ +++ ++

10-20 + + + + + + ++ + +

20-30 + + ++ ++ ++ ++ + ++

30-50 + - + - + - + + ++ + + - ++

50-70 + - + - - + - + ++ - - + -

70-100 - - - - - - - - + -

α-glu = α-glucosidase; β-Xyl = β-Xylosidase; N-acet = N-acety-β-glucosamidase, pho = acid phosphatase; β-glucoro = β-glucorosidase; β-cello = β-cellobiohydrolase; β-glu = β-glucosidase; Leu = Leucin aminopepdidase; Tyr = Tyrosin aminopepdidase; Arg = Arigin aminopepdidaseMethod: Marx et al. 2001


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ResultsResults



0-10 + + - ++ ++ - ++ - ++ - + - -

10-20 ++ + - +++ +++ + ++ - - - - - -

20-30 + + + + - ++ + - -

30-50 + - - +++ + - - - - - (2) - (2)

50-70 + - - + - + - + +

70-100 ++ - - - + - - + - - - - - (2) - (2)









Soil depth α-glu ß-Xyl N-acet ß-glucoro ß-cello ß-glu pho Leu Tyr Arg[cm]

0-10 + ++ + ++ ++ + - ++ +++ ++

10-20 + + + + + + ++ + +

20-30 + + ++ ++ ++ ++ + ++

30-50 + - + - + - + + ++ + + - ++

50-70 + - + - - + - + ++ - - + -

70-100 - - - - - - - - + -

α-glu = α-glucosidase; β-Xyl = β-Xylosidase; N-acet = N-acety-β-glucosamidase, pho = acid phosphatase; β-glucoro = β-glucorosidase; β-cello = β-cellobiohydrolase; β-glu = β-glucosidase; Leu = Leucin aminopepdidase; Tyr = Tyrosin aminopepdidase; Arg = Arigin aminopepdidaseMethod: Marx et al. 2001


Changes in SOC- quality due to continuously priming in the field by addition of TWW

– long-term effect

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Basra 0-10 cm: Enzyme activities [nmol g-1 h-1]

0

100

200

300α-glu

ß-Xyl

chit

pho

ß-celloß-glu

Leu

Tyr

Arg

FWTWW


0

20

40

60

80

100α-glu

ß-Xyl

chit

pho

ß-celloß-glu

Leu

Tyr

Arg

FWTWW


0

20

40

60

80

100α-glu

ß-Xyl

chit

pho

ß-celloß-glu

Leu

Tyr

Arg

FWTWW


0

20

40

60

80

100α-glu

ß-Xyl

chit

pho

ß-celloß-glu

Leu

Tyr

Arg

FWTWW


0

5

10

15

20

25

30

35α-glu

ß-Xyl

chit

pho

ß-celloß-glu

Leu

Tyr

Arg

FWTWW


0

2

4

6

8

10

12

14α-glu

ß-Xyl

chit

pho

ß-celloß-glu

Leu

Tyr

Arg

FWTWW

BAZRA – enzyme activities

max: 291.6 nmol g-1 h-1

max: 99.6 nmol g-1 h-1

max: 55.7 nmol g-1 h-1 max: 61.2 nmol g-1 h-1


0-10 cm 10-20 cm 20-30 cm

30-50 cm 50-70 cm 70-100 cm

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0

100

200

300α-glu

ß-Xyl

chit

pho

ß-celloß-glu

Leu

Tyr

Arg

FWTWW


0

20

40

60

80

100α-glu

ß-Xyl

chit

pho

ß-celloß-glu

Leu

Tyr

Arg

FWTWW


0

20

40

60

80

100α-glu

ß-Xyl

chit

pho

ß-celloß-glu

Leu

Tyr

Arg

FWTWW


0

20

40

60

80

100α-glu

ß-Xyl

chit

pho

ß-celloß-glu

Leu

Tyr

Arg

FWTWW


0

5

10

15

20

25

30

35α-glu

ß-Xyl

chit

pho

ß-celloß-glu

Leu

Tyr

Arg

FWTWW


0

2

4

6

8

10

12

14α-glu

ß-Xyl

chit

pho

ß-celloß-glu

Leu

Tyr

Arg

FWTWW

BAZRA – enzyme activities

max: 291.6 nmol g-1 h-1

max: 99.6 nmol g-1 h-1



0-10 cm 10-20 cm 20-30 cm

30-50 cm 50-70 cm 70-100 cm

Similar enzyme content pattern over the profile

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ResultsResults


-90

-80

-70

-60

-50

-40

-30

-20

-10

00.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50

ß-glucosidase activity [nmol h-1µg Cmic]

Soil

dept

h [c

m]

FWTWW

Bazra – soil enzyme profiles

clear differences between FW and TWW

activity increase with soil depth

-90

-80

-70

-60

-50

-40

-30

-20

-10

00.00 100.00 200.00 300.00 400.00

ß-glucosidase activity [nmol h-1g-1]

Soi

l dep

th [c

m]

FW

TWW

high correlation with microbial biomass carbon

β-glucosidasenmol h-1 g-1 dry soil nmol h-1 µg-1 Cmic

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ResultsResults & & DiscussionDiscussion


-90

-80

-70

-60

-50

-40

-30

-20

-10

00.00 0.50 1.00 1.50 2.00 2.50

Phosphatase activity [nmol h-1µg Cmic]So

il de

pth

[cm

]

FWTWW

-90

-80

-70

-60

-50

-40

-30

-20

-10

00.00 0.20 0.40 0.60 0.80 1.00

N-acetyl activity [nmol h-1µg Cmic]

Soil

dept

h [c

m]

FWTWW


-90

-80

-70

-60

-50

-40

-30

-20

-10

00.00 1.00 2.00 3.00 4.00

Leucin AP activity [nmol h-1µg Cmic]

Soil

dept

h [c

m]

FWTWW

phosphatase chitinase

leucin aminopeptidase

nmol h-1 µg-1 Cmic

similar pattern like β-glucosidase

activity increase with soil depth

maybe due to tree roots or shift in texture

Increased amounts of tree roots under TWW

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ResultsResults


-90

-80

-70

-60

-50

-40

-30

-20

-10

00.00 0.20 0.40 0.60 0.80 1.00

C:NSo

il de

pth

[cm

]

FWTWW

-90

-80

-70

-60

-50

-40

-30

-20

-10

00.00 2.00 4.00 6.00 8.00 10.00

C:P

Soil

dept

h [c

m]

FWTWW

-90

-80

-70

-60

-50

-40

-30

-20

-10

00.00 10.00 20.00 30.00 40.00

N:P

Soil

dept

h [c

m]

FWTWW

-90

-80

-70

-60

-50

-40

-30

-20

-10

00.00 0.20 0.40 0.60 0.80 1.00 1.20

Cellu : C

Soil

dept

h [c

m]

FWTWW

C : P

N : P

Bazra – soil enzyme profilesC : N

Cell : C

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ResultsResults


-90

-80

-70

-60

-50

-40

-30

-20

-10

00.00 0.20 0.40 0.60 0.80 1.00

C:NSo

il de

pth

[cm

]

FWTWW

-90

-80

-70

-60

-50

-40

-30

-20

-10

00.00 2.00 4.00 6.00 8.00 10.00

C:P

Soil

dept

h [c

m]

FWTWW

-90

-80

-70

-60

-50

-40

-30

-20

-10

00.00 10.00 20.00 30.00 40.00

N:P

Soil

dept

h [c

m]

FWTWW

-90

-80

-70

-60

-50

-40

-30

-20

-10

00.00 0.20 0.40 0.60 0.80 1.00 1.20

Cellu : C

Soil

dept

h [c

m]

FWTWW

C : P

N : P


main rooting zone?

main rooting zone?

less phosphatase necessary!


Shift?

C : N

Cellulase : C

less N-degrading enzymes necessary!

more N-degrading enzymes necessary!

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ResultsResults


-90

-80

-70

-60

-50

-40

-30

-20

-10

00.00 0.20 0.40 0.60 0.80 1.00

C:NSo

il de

pth

[cm

]

FWTWW

-90

-80

-70

-60

-50

-40

-30

-20

-10

00.00 2.00 4.00 6.00 8.00 10.00

C:P

Soil

dept

h [c

m]

FWTWW

-90

-80

-70

-60

-50

-40

-30

-20

-10

00.00 10.00 20.00 30.00 40.00

N:P

Soil

dept

h [c

m]

FWTWW

-90

-80

-70

-60

-50

-40

-30

-20

-10

00.00 0.20 0.40 0.60 0.80 1.00 1.20

Cellu : C

Soil

dept

h [c

m]

FWTWW

C : P

N : P

Bazra – soil enzyme profilesC : N

Cell : C

clear differences between FW and TWW concerning phosphatase mainly pronounced in depth P input by TWW !

Ratio C : N hydrolysing enzyme activity increasedunder TWW irrigation until the first 50 cm

shift in deeper horizons more C results in N-limitation N enzymes needed

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Akko 0-10 cm [nmol g-1 h-1]

0

100

200

300

400

500α-glu

ß-Xyl

chit

pho

ß-celloß-glu

Leu

Tyr

Arg

FWTWW


0

20

40

60

80

100α-glu

ß-Xyl

chit

pho

ß-celloß-glu

Leu

Tyr

Arg

FWTWW


0

50

100

150

200α-glu

ß-Xyl

chit

pho

ß-celloß-glu

Leu

Tyr

Arg

FWTWW


0

50

100

150

200α-glu

ß-Xyl

chit

pho

ß-celloß-glu

Leu

Tyr

Arg

FWTWW

Akko 30-50 cm [nmol-1 h-1]

0

50

100

150

200

250

300α-glu

ß-Xyl

chit

pho

ß-celloß-glu

Leu

Tyr

Arg

FWTWW


0

50

100

150

200

250

300

350α-glu

ß-Xyl

chit

pho

ß-celloß-glu

Leu

Tyr

Arg

FWTWW

Acco – enzyme activities

max: 431.4 nmol g-1 h-1

max: 271.3 nmol g-1 h-1

max:76.7 nmol g-1 h-1 max: 310.6 nmol g-1 h-1


0-10 cm 10-20 cm 20-30 cm

30-50 cm 50-70 cm 70-100 cm

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0

100

200

300

400

500α-glu

ß-Xyl

chit

pho

ß-celloß-glu

Leu

Tyr

Arg

FWTWW


0

20

40

60

80

100α-glu

ß-Xyl

chit

pho

ß-celloß-glu

Leu

Tyr

Arg

FWTWW


0

50

100

150

200α-glu

ß-Xyl

chit

pho

ß-celloß-glu

Leu

Tyr

Arg

FWTWW


0

50

100

150

200α-glu

ß-Xyl

chit

pho

ß-celloß-glu

Leu

Tyr

Arg

FWTWW

Akko 30-50 cm [nmol-1 h-1]

0

50

100

150

200

250

300α-glu

ß-Xyl

chit

pho

ß-celloß-glu

Leu

Tyr

Arg

FWTWW


0

50

100

150

200

250

300

350α-glu

ß-Xyl

chit

pho

ß-celloß-glu

Leu

Tyr

Arg

FWTWW

Acco – enzyme activities

max: 431.4 nmol g-1 h-1

max: 271.3 nmol g-1 h-1



0-10 cm 10-20 cm 20-30 cm

30-50 cm 50-70 cm 70-100 cm

Similar enzyme content pattern over the profile

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-90

-80

-70

-60

-50

-40

-30

-20

-10

00 100 200 300 400 500

ß-glucosidase activity [nmol h-1g-1 dry soil]

Soil

dept

h [c

m]

FWTWW

-90

-80

-70

-60

-50

-40

-30

-20

-10

00.00 1.00 2.00 3.00 4.00 5.00 6.00

ß-glucosidase activity [nmol h-1µg Cmic]

Soil

dept

h [c

m]

FWTWW

ResultsResults


Acco – soil enzyme profiles

clear differences between FW and TWW only in topsoil

activity increase with soil depth only under FW irrigation

β-glucosidasenmol h-1 g-1 dry soil nmol h-1 µg-1 Cmic

Distribution along the profile reflects the transportation of carbon

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-90

-80

-70

-60

-50

-40

-30

-20

-10

00.00 0.20 0.40 0.60 0.80 1.00

Cellu : C

Soil

dept

h [c

m]

FWTWW

-90

-80

-70

-60

-50

-40

-30

-20

-10

00.00 5.00 10.00 15.00

N:P

Soil

dept

h [c

m]

FWTWW

-90

-80

-70

-60

-50

-40

-30

-20

-10

00.00 5.00 10.00 15.00 20.00 25.00

C:P

Soil

dept

h [c

m]

FWTWW

-90

-80

-70

-60

-50

-40

-30

-20

-10

00.00 0.50 1.00 1.50 2.00

C:NSo

il de

pth

[cm

]

FWTWW

ResultsResults & & DiscussionDiscussionC : P

N : P

Acco – soil enzyme profilesC : N

Cell : C

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-90

-80

-70

-60

-50

-40

-30

-20

-10

00.00 0.20 0.40 0.60 0.80 1.00

Cellu : C

Soil

dept

h [c

m]

FWTWW

-90

-80

-70

-60

-50

-40

-30

-20

-10

00.00 5.00 10.00 15.00

N:P

Soil

dept

h [c

m]

FWTWW

-90

-80

-70

-60

-50

-40

-30

-20

-10

00.00 5.00 10.00 15.00 20.00 25.00

C:P

Soil

dept

h [c

m]

FWTWW

-90

-80

-70

-60

-50

-40

-30

-20

-10

00.00 0.50 1.00 1.50 2.00

C:NSo

il de

pth

[cm

]

FWTWW


N : P

Acco – soil enzyme profiles

main rooting zone?


main rooting zone?


Shift?

C : N

Cell : C

less N-degrading enzymes necessary!

more N-degrading enzymes necessary!

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-90

-80

-70

-60

-50

-40

-30

-20

-10

00.00 0.20 0.40 0.60 0.80 1.00

Cellu : C

Soil

dept

h [c

m]

FWTWW

-90

-80

-70

-60

-50

-40

-30

-20

-10

00.00 5.00 10.00 15.00

N:P

Soil

dept

h [c

m]

FWTWW

-90

-80

-70

-60

-50

-40

-30

-20

-10

00.00 5.00 10.00 15.00 20.00 25.00

C:P

Soil

dept

h [c

m]

FWTWW

-90

-80

-70

-60

-50

-40

-30

-20

-10

00.00 0.50 1.00 1.50 2.00

C:NSo

il de

pth

[cm

]

FWTWW


N : P


Cell : C



shift in deeper horizons

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-90

-80

-70

-60

-50

-40

-30

-20

-10

00.00 0.20 0.40 0.60 0.80 1.00

Cellu : C

Soil

dept

h [c

m]

FWTWW

-90

-80

-70

-60

-50

-40

-30

-20

-10

00.00 5.00 10.00 15.00

N:P

Soil

dept

h [c

m]

FWTWW

-90

-80

-70

-60

-50

-40

-30

-20

-10

00.00 5.00 10.00 15.00 20.00 25.00

C:P

Soil

dept

h [c

m]

FWTWW

-90

-80

-70

-60

-50

-40

-30

-20

-10

00.00 0.50 1.00 1.50 2.00

C:NSo

il de

pth

[cm

]

FWTWW


N : P


Cell : C



shift in deeper horizonsSame result as found for Bazra! even more pronounced,

maybe due to higher clay contents?

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0.2

0.6

1.0

1.4

1.8

2.2

0-10

10-2

0

20-3

0

30-5

0

50-7

0

70-1

00

0-10

10-2

0

20-3

0

30-5

0

50-7

0

70-1

00

ln(B

G):l

n(NAG

+LAP)

C:N

FWTWW

ResultsResults Nutrient aquisation activity (Sinsabaugh et al. 2008)

0.2

0.6

1.0

1.4

1.8

2.2

0-10

10-2

0

20-3

0

30-5

0

50-7

0

70-1

00

0-10

10-2

0

20-3

0

30-5

0

50-7

0

70-1

00

ln(B

G):l

n(AP)

C:P

FWTWWC:P

C:N

Jüschke & Marschner(calculated on the basis of g dry soil)

Bazra Acco

Bazra Acco

Ln(BG):Ln(AP)

Ln(BG):Ln(NAG+LAP)

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0.2

0.6

1.0

1.4

1.8

2.2

0-10

10-2

0

20-3

0

30-5

0

50-7

0

70-1

00

0-10

10-2

0

20-3

0

30-5

0

50-7

0

70-1

00

ln(B

G):l

n(NAG

+LAP)

C:N

FWTWW

ResultsResults & & DiscussionDiscussion Nutrient aquisation activity (Sinsabaugh et al. 2008)

0.2

0.6

1.0

1.4

1.8

2.2

0-10

10-2

0

20-3

0

30-5

0

50-7

0

70-1

00

0-10

10-2

0

20-3

0

30-5

0

50-7

0

70-1

00

ln(B

G):l

n(AP)

C:P

FWTWWC:P

C:N

Jüschke & Marschner(calculated on the basis of g dry soil)

Bazra Acco

If significant differences occur, then ratios arehigher under TWW irrigation!

Nutrient aquisation activity after Sinsabaugh et al. (2008)was compared with these dataData fit in the concept

Sinsabaugh et al. (2008) FW TWWC:N 1.02 ± 0.2 1.11 ± 0.13 1.16 ± 0.13 C:P 0.95 ± 0.15 0.98 ± 0.12 1.03 ± 0.05

Trend of higher ratio, but not significant !

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SummarySummary and and ConclusionConclusion


Higher total enzyme activity = higher degradation of SOC

…before it´s open for discussion …

Transport of carbon down the soil profiles, which is then used as source for microorganisms

Continously triggered priming effects resulted in strongerdecrease of SOC

Decreased phosphatase activity in the subsoil horizons

P supply by the TWW is enough, less phosphatase necessary

Clay-humus-complexes3-dimensional network

active enzymes incorporated (Paul and Mc Laren, 1975)

Clay minerals as possible drivers for enzyme activity (clay bind proteins)

not masked by C input in the subsoil

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Thank you for attention

Picture: drip irrigation system - Elisabeth Jüschke 2004

Acknowledgements

- Our cooperation partners: Yona Chen, Yitzhak Hadar (Israel)Jamal Safi (Gaza Strip)

- My recent workplace, the MPI for Biogeochemistry in Jena, Germany

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activity of exoenzymes in treated wastewater irrigated soils · jüschke & marschner activity...

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