Symposium Jubileum ILST

Parameters of biogasproduction: the microbial connection

Symposium Jubileum ILST “50 jaar laboratoriumonderwijs in Groningen” 28 april 2015

05/10/15

Dr. Emile Apol, Lecturer Chemistry, Chemical Technology and Biomedics. Specialism:

Mathematical Modelling

Dr. Folkert Faber, Senior Lecturer, Researcher Biological and Medical Research

Marina Gil Lopez, MSc Research Assistant

Dr. Ir. J.P Nap, lector van o.a

biomassa-transformatie binnen het Energiekenniscentrum

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8 05/10/15 1.  Hydrolysis 2.  Acidogenesis 3.  Acetogenesis 4.  Methanogenesis

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To solve this problem we will use an internal

(external) standard (IPC), which, after isolation of

DNA, to all the samples is added.

In this way we will create a housekeeping-gene

to which we will calculate all samples relative to

this known amount

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Another technique

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aq-ma

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Other PCR-device

CFX96/biorad

Experimental Design:

•  2 Infors, 10 liter labscale bioreactors, F1 and F2

•  Filled with manure (from cows of (another) highly valued

colleague)

•  F1: biomass Hay (untreated) •  F2: Kitchenwaste

•  Hydrolic Retention-Time (HRT) : 30 days

•  Organic Loading Rate (OLR): 3 kg/m3_/day

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Results: methane potential AMPTSII

Biomass Average ml (n=2) Biogas (ml) Biogas (l/kg)

Hay (6,2 grams OL) 2933 +/-116 1855 299 KW (5,2 grams OL) 3647 +/- 40 2569 494 PreHay (6,2 grams OL) 2538 +/- 17 1460 236 Inoculum (blanc) 1078 +/- 32 - -

Table 1: maximum biogaspotential of Hay, PreHay and Kitchen-Waste

*Marina Gil Lopez *Folkert Faber

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aq-ma

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: internal /external PCR control

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Normalizer assay:

Target assay:

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The primer Tm should be the same for all

primers used in the multiplex reaction.

Same results would be obtained if the

reactions were performed individually.

They compete for the same reagents.

It is important that this competition be

minimized.

Both assays are amplified in the same tube:

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√

√

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Micro-organism Ra9o F2/F1

(day=0) Ra9o F2/F1 (day=30) Ra9o F2/F1 (day=60)

Archea (Arc) _{0,26 +/- 0,06 0,96 +/- 0,23 7,03 +/- 1,35} Methanosarcinales (MSL) _{0,31 +/- 0,05 1,66 +/- 0,27 0,82 +/- 0,16} Methanomicrobiales (MMB) _{0,41 +/- 0,07 3,05 +/- 0,80 22,06 +/- 4,57}

(preliminary) results: community-study

MSL :CH₃COOH → CH₄ + CO₂ MMB: CO₂ + 4 H₂ → CH₄ + 2H₂O

0.263 0.307 0.41 0.963 1.667 3.055 7.032 0.824 22.063

DAY 0 DAY 30 DAY 60

community-study

F2 (KW) versus F1(Hay)

05/10/15 Time (days) MSL MMB T= 0 _{0,86 < 1.17 < 1,59 1,13 < 1,56 <2,15} T= 30 _{1,25 < 1,73 < 2,40 2,10 < 3,17 <4,79} T= 60 _{1,27 < 1,81 <2,58 2,15 < 3,06 <4,34} Table 2: The raTo’s of MSL (F2/F1) to ARC and the raTo of MMB (F2/F1) to ARC: NORMALIZED to ARC 24

Optimising Processes for the Stable Operation of Food Waste Digestion, 2011, School of Civil

Engineering and the Environment, University of Southampton. Dr Yue Zhang, Dr Mark Walker and Professor Charles J Banks

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This study: If we compare the biomass fermentation of Hay with Kitchen Waste, we see an increase in the

amount Methanomicrobiales (MMB) : the

hydrogentrophic Methanogen. (CO₂ + 4 H₂ → CH₄ + 2H₂O) Several studies show that the degradation of Kitchen Waste into methane, MMB plays a (much) bigger role than MSL

It seems that the 'PCR tool' works and we are now ready do new experiments to investigate the role of

various process parameters, and different biomasses on the methanogenic community in biogasproduction.

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Future Plans

Further and better ‘Modelling’ the community with Taqman and Metagenomics

Optimization of the Taqman assay (more groups)

Metagenomics (Martijn Herber en Ronald Wedema BFV) Enrichment study with addition of

1.) Acetate (community-shift towards MSL)

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Instituut voor Life Science & Technology