Anaerobic fermentation of protein-rich substrates

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Anaerobic fermentation of protein-rich substrates

Etelka Kovács

SZTE TTIK Department of Biotechnology Straub-days

23/05/2012

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Renewable energy sources

Non limited Limited

Wind power Solar-energy Hydropower

Geothermal energy

Soil Biomass

Biogas

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Production of biogas

Hydrolyzing microbes Polymer degradation

Acetogenic bacteria Organic acids and H ₂

Methanogens

CH ₄ and CO ₂

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Degradation of protein

Protein

Amino acids

Organic acids,

NH ₃ + CO ₂ + H ₂ + H ₂ S Proteases

Stickland-reaction or

Reductive deamination

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Substrates, volume - system

• Casein, blood, meat extract, kitchen waste

• Batch, 5 L and 50 L continuous

fermentors

• HPLC

• GC – gas yield

• Enzyme activity

• pH

• Redox potential

• NH ₄ ⁺

• Next generation sequencing

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SOLiD V4, SOLiD 5500XL

Read length: 50 bases (single reads and paired end reads)

Generated tags (reads): 1000 Million /run Throughput: 50 Gbase/slide/run

(10 000x coverage on 5 Mb bact. genome)

Highest accuracy (no homopolymer issue, two-

base encoding)

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Casein adaptation 5 L

0.0 0.5 1.0 1.5 2.0 2.5

Bio gas-yield (l)/ 1 g oTS

Casein (g)

90001900ral 90011900ral 90021900ral 90031900ral 90041900ral 90051900ral 90061900ral 90071900ral 90081900ral 90091900ral

190001900ral 190011900ral 190021900ral 190031900ral 190041900ral 190051900ral 190061900ral 190071900ral 190081900ral 190091900ral 190010190… 190011190… 190012190… 190013190… 190014190… 190015190… 190016190…

Organic acid concentration (g/l)

Time (week)

Acetic acid (mg/ml) Propionic acid (mg/ml) Izobutiric acid

Butiric acid

Izovaleric acid (mg/ml)

0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40

Absorbance

Time (week)

Enzyme-activity

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Bacilli Clostridia Erysipelotrichi 0

20 40 60 80 100

Bacteroidetes Firmicutes Proteobacteria

Abundance (%) Casein starting

Casein half-time Casein dying

Actinobacteria Chloroflexi Deinococcus- Thermus Dictyoglomi Fusobacteria Nitrospirae Planctomycetes Spirochaetes Synergistetes Tenericutes

0 10 20 30 40 50 60 70 80 90 100

Bacteroidia Cytophagia Flavobacteria

Abundance(%)

Bact. Cyto. Flavo.

β-proteob. γ-proteob. δ-proteob.

0 10 20 30 40 50 60 70 80 90 100

Bacillales Lactobacillales

Abundance(%)

Clostridiales Halanaerobiales Natranaerobiales Thermoanaerob.

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0 10 20 30 40 50 60 70 80 90 100

Clostridiales Halanaerobiales Natranaerobiales Thermoanaerob.

Abundance(%)

0 20 40 60 80 100

Firmicutes Proteobacteria

Abundance(%)

10 0 20 30 40 50 60 70 80 90 100

Clostridia Erysipelotrichi

Abundance (%)

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Kitchen waste vs. meat-extract (C:N)

190001900ral 190001900ral 190001900ral 190001900ral 190001900ral 190001900ral 190001900ral 190001900ral 190001900ral

1-2. 3-4. 5-6. 7-8. 9-10.

Bio gas-yield (NL/1 g oTS)

Time (week)

Húsliszt

Konyhai hulladék Meat-extract Kitchen waste

Archaea

(%)

Bacteria

(%)

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190001900ral 1900101900ral 1900201900ral 1900301900ral 190091900ral 1900191900ral

Abundance(%)

Bact. Cyto. Flavo. Bacilli Clostridia Synergistia Thermotogae (class)

Abundance(%)

Bact. Cyto. Flavo.

0 10 20 30 40 50 60

Bacteroidetes

Abundance(%)

Firmicutes Synergistetes

Thermotogae

Konyhai hulladék Húsliszt

Kitchen waste Meat extract

Clostridiales Halanaerobiales Thermoanaerob.

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Bacilli Clostridia

Abundance(%)

0 10 20 30 40 50 60

Firmicutes

Abundance(%)

Clostridiales Halanaerobiales Thermoanaerob.

Abundance(%)

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Summary

• Microbial communities can be adapted and engineered

• Excellent biogas yields from protein-rich substrates

• Monitoring microbes during fermentation help

to improve the process

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Acknowledgements

Prof. Dr. Kornél L. Kovács Dr. Gábor Rákhely

Dr. Zoltán Bagi Dr. Gergő Maróti

Norbert Ács Roland Wirth

All members of Dept. of Biotechnology

TÁMOP-4.2.1/B-09/1/KONV-2010-0005, Baross_ALGOLABH, and TÁMOP

4.2.2/B-10/1-2010-0012

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Anaerobic fermentation of protein-rich substrates