Variation in the solubilization of

nitrogenous compounds in wheat straw

by different white-rot fungi

Nazri Nayan1, Anton Sonnenberg2, John Cone1, Wouter Hendriks1

1 Animal Nutrition Group, Wageningen University; 2 Plant Breeding, Wageningen University

69th EAAP Annual Meeting

Scholarship Co-sponsored by

2

Background

Food

FuelFeed

Increasing human

population

Demand for animal-

based products

Sustainable animal

production

Green energy

Government subsidies

Food security

Profitability

Increase of feed price Depletion of fossil fuels

Abundances of biomass

Sustainability issues

3

Background

Food

Feed

Demand for animal-

based products

Sustainable animal

production

Food security

Increase of feed price

Abundances of biomass

Sustainability issuesgrain

straw

Background Unlocking the lignin-carbohydrates complex

4

Cellulose46.1 to 50.0%

Hemicellulose26.0 to 33.8%

Lignin *5.6 to 9.2%

Protein1.1 to 3.3%

* Acid detergent lignin; Data obtained from 3 independent experiments

Background

5

Unlocking the lignin-carbohydrates complex

Biological

Physical + Chemical

Hendriks et al., Bioresour. (2009); Hatakka & Hammel, The Mycota (2010)

Background

6

Unlocking the lignin-carbohydrates complex

Lignin-degrading

enzymes

Glycoside

hydrolases (GHs)Proteases

LiP MnP LacVP

Cell Hemi

Hendriks et al., Bioresour. (2009); Hatakka & Hammel, The Mycota (2010)

Background

7

Unlocking the lignin-carbohydrates complex

Lignin

Available

carbohydrates

In vitro Degradability

Tuyen et al., Bioresour. (2012); van Kuijk et al., J. Anim. Sci. Biotechnol (2016)

Protein

availability?

8

Background Nitrogen metabolism by fungi

[1] Tudzynski, Front. Microbiol. (2014); [2] Leatham & Kent , FEMS Microbiol. Let. (1983); [3] Van Kuijk et al., Anim. Feed Sci. Technol. (2015);

[4] Dorado et al. App. Microbiol. Biotech. (2001)

Assimilate N from various sources [1]Various effects on ligninolytic enzymes [2]No effect of urea supplementation [3]N removal improves selectivity [4]

Background Previous selection for high potential fungi

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Lentinula edodes

Shiitake mushroom

Pleurotus eryngiiKing oyster/trumpet mushroom

Ceriporiopsis subvermispora

No fruiting body formation

12

10

10

Sele

cte

d b

ase

d o

n I

VG

PNayan et al., J. Appl. Microbiol. (2018)

A

B1

B2

B3

C

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Objective

Fractionation of crude protein [1] In vitro N availability [2]

[1] Sniffen et al. J. Anim. Sci. (1992); [2] Cone et al., Anim. Feed Sci. Technol. (2009)

Fungal preparation

2-3 weeks 4-5 weeks

Agar plate Spawn

Wheat straw preparation

Soaking 3 days

Autoclave 1 hour

Inoculate wheat straw

with spawn (10% dry

weight)

7 weeks

Treatment Analyses

• Fractionation of

CP

• In vitro

assessment of N

availability

• Amino acids

Methodology

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Methodology

12

Fractionation of crude protein

Sniffen et al. J. Anim. Sci. (1992)

Total Protein

Buffer Soluble P Buffer Insoluble P

NDIP

ADIP

NPN True Protein

A B1 B2 B3 C

Methodology

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In vitro assessment of N availability

Cone et al., Anim. Feed Sci. Technol. (2009)

N-Free buffer

Rapidly soluble carbohydrates

Pre-incubation (5 h)

IVG

P (m

l)

Incubation time (h)

Methodology

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In vitro assessment of N availability

Cone et al., Anim. Feed Sci. Technol. (2009)

N-Free buffer

Rapidly soluble carbohydrates

Pre-incubation (5 h) In vitro gas production (72 h)

Pre-weighed samples containing 5 mg N

60 ml of pre-incubated rumen fluid + remaining carbohydrates

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Results Mass balances

0

20

40

60

80

100

Control CS1 CS12 PE3 PE6 LE8 LE10

Am

ou

nt

(g/1

00

g s

tart

ing

OM

)

Fungal strains/species

Cellulose Hemicellulose Lignin Crude protein Unaccounted OM OM degraded

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Results Mass balances

OM degraded ~7.3%

Unaccounted OM 2.9 times higher

CP 28.2%

Lignin 52.2%

Hemicellulose 49.7%

Cellulose 6.0%

Control vs. CS1-treated wheat straw

Control CS1

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Results Fractionation of CP

0

20

40

60

80

100

Control CS1 CS12 PE3 PE6 LE8 LE10

Fra

cti

on

s (%

CP

)

Fungal strains/species

A B1 B2 B3 C

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Results Fractionation of CP

0

20

40

60

80

100

Control CS1 CS12 PE3 PE6 LE8 LE10

Fra

cti

on

s (%

CP

)

Fungal strains/species

A B1 B2 B3 C

0

20

40

60

80

100

Control CS1 CS12 PE3 PE6 LE8 LE10

Fra

cti

on

s (%

CP

)

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Results Fractionation of CP

50.2a 21.4b 25.6b Fraction C

Expressed as %CP. Values with different superscripts are significantly (P < 0.05) different

0

20

40

60

80

100

Control CS1 CS12 PE3 PE6 LE8 LE10

Fra

cti

on

s (%

CP

)

20

Results Fractionation of CP

10.5a 20.6b 37.7c Fraction A

Expressed as %CP. Values with different superscripts are significantly (P < 0.05) different

0

20

40

60

80

100

Control CS1 CS12 PE3 PE6 LE8 LE10

Fra

cti

on

s (%

CP

)

21

Results Fractionation of CP

15.2a 36.1b 20.2ac Fraction B2

Expressed as %CP. Values with different superscripts are significantly (P < 0.05) different

40

50

60

70

80

90

0 20 40 60IV

GP

N(m

l/5 m

g N

)Fraction C (% CP)

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Results In vitro gas production (IVGPN)

30

40

50

60

70

80

90

100

Control CS1 CS12 PE3 PE6 LE8 LE10

IVG

PN

(ml/

5 m

g N

)

Fungal strains/species

a

a

a

b

b

b

b

r = -0.46

P = 0.002

vs. Fraction A

vs. Fraction C

Values with different superscripts are significantly (P < 0.05) different

40

50

60

70

80

90

0 10 20 30 40

IVG

PN

( m

l/5 m

g N

)

Fraction A (% CP)

r = 0.79

P < 0.001

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Take-home messages

Fungi are varied in their capabilities of solubilizing N in the

wheat straw

P. Eryngii and L. edodes strains increase the N availability in

the rumen; but not C. subvermispora strains

Fractionation of protein and in vitro evaluation of protein

fermentation in rumen fluid are reliable parameters in

assessing protein solubilization by different fungal strains.

24

Published as: Nayan et al. (2018).

Variation in the

solubilization of crude

protein in wheat straw by

different white-rot fungi

Anim. Feed Sci. Technol., 242:135-143

T e r i m a K a s i h

Thank you