Plant A and Plant B: two agricultural biogas plants
Results
| Item | Plant A (pig manure) | Plant B (maize silage) | Status A | Status B |
|---|---|---|---|---|
| Methanogens (all routes) | 7 genomes, 4.9 % | 5 genomes, 1.5 % | ok | ok |
| Acetate route (acetate-consuming methanogens) | Methanothrix soehngenii, 3.7 % | Methanosarcina flavescens, 0.49 % | ok | note |
| H₂/CO₂ route (hydrogen-consuming methanogens) | 3 genomes, 0.67 % | 5 genomes, 1.5 % | note | ok |
| Methyl route (methyl-consuming methanogens) | 2 genomes, 0.40 % | 1 genome, 0.49 % | ok | ok |
| Cellulose breakdown (genomes with enzymes anchored outside the cell) | 8 genomes, 3.4 % | 6 genomes, 1.9 % | ok | ok |
| Protein breakdown (genomes with enzymes anchored outside the cell) | 5 genomes, 5.6 % | 10 genomes, 3.7 % | ok | ok |
| Propionate pathway (genomes carrying it) | 31 genomes, 37.1 % | 25 genomes, 20.4 % | ok | ok |
| Ammonia release from ethanolamine (genomes carrying the pathway) | 5 genomes, 3.6 % | 1 genome, 0.45 % | note | ok |
| Butyrate oxidation | — | — | not assessed | not assessed |
| Ethanol fermentation | — | — | not assessed | not assessed |
| Lactate fermentation | — | — | not assessed | not assessed |
| Direct interspecies electron transfer | — | — | not assessed | not assessed |
| Energy conservation | — | — | not assessed | not assessed |
Each value gives the number of genomes and their share of reads. Share of reads = the part of the mapped reads that the genomes make up. ok = nothing unusual. note = thin or present, not a fault by itself: the text below names the measurement to check. attention = a route is missing or very weak. not assessed = no reliable marker, not zero.
Findings
- Both plants have a complete methane community. Both routes are present. This is the normal state.
- Plant A: one genome, Methanothrix soehngenii, carries all of the acetate route. It is abundant. It is sensitive to free ammonia and to sudden load changes. The feed has 3.8 g/L NH₄⁺-N.
- Plant A: 5 genomes (3.6 %) can release ammonia from ethanolamine in manure.
- Plant B: one genome, Methanosarcina flavescens, carries all of the acetate route. It is at low abundance (0.49 %). It tolerates ammonia and acid spikes. It needs higher acetate levels than Methanothrix to work.
- Plant B has dedicated cellulose degraders. Two genomes from the Clostridium thermocellum group (Acetivibrionaceae) carry all 6 enzyme families tested in anchored form.
- Neither plant shows the pattern that gets an attention flag: H₂/CO₂ methanogens present and no acetate consumer.
Where each plant is weak
In both plants the acetate route rests on one genome. The difference is which organism it is and how much of it there is.
| Plant A | Plant B | |
|---|---|---|
| Acetate consumer | Methanothrix soehngenii | Methanosarcina flavescens |
| Share of reads | 3.7 %, abundant | 0.49 %, scarce |
| Tolerance | sensitive to free ammonia and sudden load changes | tolerates ammonia and acid peaks, and needs more acetate to work |
| Routes it can run | acetate specialist | all three routes, a generalist |
| Methanogens in total | 4.9 % | 1.5 % |
| Ammonia release from ethanolamine (genomes carrying the pathway) | 5 genomes, 3.6 % | 1 genome, 0.45 % |
- Plant A: a strong but sensitive acetate route. An abundant acetate specialist lives with ammonia-rich feed and more ammonia-releasing genomes. The main risk is rising ammonia.
- Plant B: an ammonia-tolerant but thin acetate route. The literature gives this genus a high tolerance, but the genome is scarce here. The main risk is a load increase that is faster than the community can take up acetate.
Protein breakdown also releases ammonia. It is similar in both plants (51.5 % and 56 % of reads). Most of the ammonia comes with the feed. Ammonia release capacity is not a measurement of ammonia. Read it together with your NH₄⁺-N data. The two plants come from two different studies. The literature gives the tolerance and the acetate affinity of the two genera. We did not measure them in these samples. See Jetten et al. 1992, FEMS Microbiol Rev 8:181, and De Vrieze et al. 2012, Bioresour Technol 112:1.
Suggestions
These suggestions come from the genes that the community carries. They are not operating instructions. The plant operator has the process data and makes the decisions on feed, load and additives. Magrittr OÜ is not responsible for those decisions or their results.
Fixed rules make these suggestions from the result tables. The cheapest action comes first. Each line shows the finding that caused it.
Plant A
- Watch propionate. Bacteria break it down only while hydrogen and acetate stay low, and methanogens keep hydrogen low. This report found few hydrogen-consuming methanogens. (Schink 1997; Schnürer & Jarvis 2018)
Why: 3 genomes carry the H₂/CO₂ route (0.67 %), including any that also use acetate. Acetate route: Methanothrix soehngenii, 3.7 % of mapped reads. 31 genomes carry propionate-pathway genes, and DNA does not show the direction. - Compare these results with your FOS/TAC (VOA/TAC), NH₄⁺-N, propionate, pH and temperature from the same weeks, and with this reactor's own history. (Drosg 2013)
Why: Genes show capacity. The process data show what the reactor does now. FOS/TAC values differ between plants. - Take the next sample in 3 months, or sooner after a change of feed or an upset, at the same point and before feeding.
Why: One sample is a baseline. The second sample shows what moved. The interval and the time before feeding are our protocol, kept the same so that two samples compare. - The genes support only small steps in the loading rate. (FNR 2010)
Why: The whole acetate route rests on one genome, Methanothrix soehngenii (3.7 %). - More protein in the feed, such as manure or slaughter waste, raises the ammonia load this route is sensitive to. Only a slow rise gives the community time to adapt. NH₄⁺-N, pH and temperature show where the reactor stands. (De Vrieze et al. 2012; Schnürer & Jarvis 2018)
Why: The acetate route rests on Methanothrix, of the family Methanotrichaceae. This family is sensitive to free ammonia. 5 genomes (3.6 %) can also release ammonia from ethanolamine. - This assay does not measure trace elements (Fe, Co, Ni, Se, Mo, W). With a high manure share a lack is rare: this check rules a cause out. Handbooks first rule out overload and ammonia, then measure before any addition. An excess inhibits the process and can put the digestate over the fertiliser limit. (Banks et al. 2012; Demirel & Scherer 2011; Drosg 2013; FNR 2010)
Why: H₂/CO₂ route: 0.67 %. Acetate route: Methanothrix soehngenii, 3.7 % of mapped reads. Studies link a lack of selenium and cobalt to propionate accumulation.
Plant B
- Compare these results with your FOS/TAC (VOA/TAC), NH₄⁺-N, propionate, pH and temperature from the same weeks, and with this reactor's own history. (Drosg 2013)
Why: Genes show capacity. The process data show what the reactor does now. FOS/TAC values differ between plants. - Take the next sample in 3 months, or sooner after a change of feed or an upset, at the same point and before feeding.
Why: One sample is a baseline. The second sample shows what moved. The interval and the time before feeding are our protocol, kept the same so that two samples compare. - The genes do not support a higher loading rate before the next sample. (Schnürer & Jarvis 2018)
Why: The acetate route is thin: Methanosarcina flavescens, 0.49 % of mapped reads. 2 genomes carry the Wood–Ljungdahl pathway, so bacteria may also oxidise the acetate. That route is slower and may need a long retention time.
Samples
| Plant A | Plant B | |
|---|---|---|
| Feed | pig manure | 69 % maize silage, 19 % cattle manure, 12 % chicken manure |
| Feed total solids | 37 g/L | 293 g/kg |
| Feed NH₄⁺-N | 3.8 g/L | 1.7 g/kg |
| Feed acetate | 12.1 g/L | 1.9 g/kg |
| Genomes scored (share of reads) | 137 (84.6 %) | 137 (87.4 %) |
| Source metagenome | PRJNA356200 | PRJNA301928 |
Feed values are the catalogue authors' calculated feed compositions. About 85 % of the reads in each sample map to the catalogue. The rest is reported as not scored, never as absent.
Methanogens
Plant A · 7 methanogens · 4.9 % of mapped reads
Plant B · 5 methanogens · 1.5 % of mapped reads
A route is reported only if a genome has the mcrA gene and the genes of that route. For the acetate route the genome must also belong to a known acetate-consuming group.
Plant A: one abundant specialist does the acetate work. Plant B: one low-abundance generalist does it, with help from partner bacteria that oxidise acetate to H₂ and CO₂. Both plants depend on a single species for the acetate route. The risks differ. Plant A: free ammonia and shock loads. Plant B: loss of the partner bacteria.
Breakdown capacity
A genome can have an enzyme gene while the enzyme never leaves the cell. We look for a domain on the same protein that anchors the enzyme to the cell surface or to the fibre (dockerin, CBM, S-layer). Such enzymes are built to work outside the cell. Enzymes released without such a domain are not detected, so these counts are a lower bound.
| Substrate | Plant A: carry → anchored | Plant B: carry → anchored |
|---|---|---|
| Cellulose, hemicellulose, starch (6 families) | 69 genomes → 8 anchored, 3.4 % of reads | 70 genomes → 6 anchored, 1.9 % of reads |
| Proteins (1 family) | 73 genomes → 5 anchored, 5.6 % | 89 genomes → 10 anchored, 3.7 % |
| Lipids (1 family) | 13 genomes → 0 anchored | 10 genomes → 0 anchored |
| Propionate pathway (carry only) | 31 genomes, 37 % | 25 genomes, 20 % |
| Ammonia release from ethanolamine (carry only) | 5 genomes, 3.6 % | 1 genome, 0.5 % |
Plant B has classic cellulose degraders. Plant A has single enzyme families spread over many genomes. This is the expected difference between a fibre-fed and a slurry-fed reactor. Lipases leave the cell by a different export path that this test does not see. A zero for lipids is normal.
The most abundant genome in Plant A belongs to the Smithellaceae (7.3 %), the family of Smithella. Its relatives oxidise propionate together with hydrogen-consuming methanogens. This fits a manure digester with a high feed acetate.
Not assessed
Five of the 20 panel modules have no reliable marker. This report shows them as not assessed, never as zero. Glycolysis is not scored either, for a different reason: it is assumed present in every genome. That is not "we could not tell". It is "this would tell you nothing". Two scored modules carry a caveat, below.
| Module | Why it is not scored | What it would have told you |
|---|---|---|
| Butyrate oxidation (syntrophic) | No licence-clean detector for bcd and crt: the public HMM families are superfamily-wide and would match ordinary fatty-acid metabolism. | Whether butyrate can be cleared by partner bacteria. |
| Ethanol fermentation | Only accessory-tier markers are curated, and scoring reads the core tier. | Which minor acid routes feed the methanogens. |
| Lactate fermentation | Only accessory-tier markers are curated, and scoring reads the core tier. | Which minor acid routes feed the methanogens. |
| Direct interspecies electron transfer | No core tier defined yet. | Whether conductive partnerships between species are present. |
| Energy conservation (ATP synthase, Ech, Fpo) | No core tier defined yet. | Whether energy-saving complexes are present. |
| Glycolysis | Assumed present by design: central metabolism, not a discriminating marker. | Nothing a digester operator would act on. |
DNA also cannot answer these questions. It does not show activity or rate. It does not show enzymes that leave the cell by the general (Sec) pathway. It does not show which methylated substrate the methyl route uses. It does not cover the 15 % of reads that the report did not score. Taxonomy is from GTDB-Tk, as on a client sample.
Method
Genomes and abundances: Campanaro et al. 2020, Biotechnol Biofuels 13:25 (CC BY), NCBI PRJNA602310. Genomes at ≥ 0.1 % of reads in each sample. Taxonomy: GTDB-Tk 2.7.1, GTDB release 232. Panel: digestome v0, with 86 marker genes in 20 modules. The HMMs come from NCBIfam and Pfam. The reactions come from Rhea. The panel does not use KEGG, MetaCyc, CAZy, dbCAN, MEROPS or SignalP. The panel was tested on 1,401 digester genomes and on 3,043 GTDB species. It gave no false methanogenesis route. The scripts in the digestome repository make every number again from the public inputs. Literature behind the suggestions. Schink B (1997) Microbiol Mol Biol Rev 61:262. De Vrieze J et al. (2012) Bioresour Technol 112:1. Banks CJ et al. (2012) Bioresour Technol 104:127. Demirel B, Scherer P (2011) Biomass Bioenergy 35:992. Handbooks (they support the lever and its direction; none bases a decision on the community): Drosg B (2013) Process monitoring in biogas plants. IEA Bioenergy Task 37. FNR (2010) Guide to Biogas: From production to use. 5th ed., English. Schnürer A, Jarvis Å (2018) Microbiology of the biogas process. SLU.