Plant BP3: the same reactor sampled twice, one year apart
Results
| Item | T1 (first) | T2 (second) | Change | Status |
|---|---|---|---|---|
| Methanogens (all routes) | 6 genomes, 8.0 % | 7 genomes, 4.2 % | decreased | note |
| Acetate route (acetate-consuming methanogens) | none | Methanosarcina flavescens, 0.14 % | detected at T2 | attention |
| H₂/CO₂ route (hydrogen-consuming methanogens) | 5 genomes, 7.9 % | 7 genomes, 4.2 % | decreased | note |
| Methyl route (methyl-consuming methanogens) | 1 genome, 0.18 % | 1 genome, 0.14 % | no change | ok |
| Cellulose breakdown (genomes carrying the enzymes) | 22 genomes, 14.1 % | 18 genomes, 8.2 % | decreased | note |
| Protein breakdown (genomes carrying the enzymes) | 71 genomes, 41.4 % | 71 genomes, 40.4 % | no change | ok |
| Propionate pathway (genomes carrying it) | 19 genomes, 11.0 % | 14 genomes, 12.0 % | no change | ok |
| Ammonia release from ethanolamine (genomes carrying the pathway) | 2 genomes, 0.84 % | 1 genome, 0.12 % | decreased | ok |
| Butyrate oxidation | — | — | — | not assessed |
| Ethanol fermentation | — | — | — | not assessed |
| Lactate fermentation | — | — | — | not assessed |
| Direct interspecies electron transfer | — | — | — | not assessed |
| Energy conservation | — | — | — | 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. This report shows a change only when it is at least 1 percentage point and at least 1.5-fold. Genomes below 0.1 % of reads are not in this report. Therefore "detected at T2" means: below that cutoff at T1, or absent at T1. ok = nothing unusual. note = thin, present or changed, not a fault by itself: the one-page summary names the measurement to check. attention = a route is missing or very weak. not assessed = no reliable marker, not zero.
Findings
- At T1 no genome carried the acetate route. At T2 one genome carries it: Methanosarcina flavescens, at 0.14 % of mapped reads. This is the lowest share that this report can show.
- The H₂/CO₂ methanogens fell from 7.9 % to 4.2 % of mapped reads. The largest one, Methanoculleus, fell from 3.8 % to 0.6 %. But the balance between the routes did not change. The H₂/CO₂ route is approximately 99 % of the mcrA carriers at T1, and approximately 100 % at T2. What changed is the share of the methanogens in the whole community. Within that route the genus composition did change: Methanoculleus went from about half of the methanogens to about one seventh. A different DNA extraction can also change the genus shares. Read this together with the note on the two studies.
- Cellulose breakdown carriers fell from 22 genomes to 18 genomes (14.1 % to 8.2 % of mapped reads). Protein breakdown carriers did not change: 71 genomes at both times. Bacterial shares change when the whole community changes. Read them together with the feed data.
- In the same period the feed ammonium increased from 1.06 g/kg to 1.25 g/kg. The feed acetate increased from 3.4 g/kg to 4.0 g/kg.
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.
- 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. Here the second sample showed one acetate-route genome just above the reporting cutoff, and a smaller share of methanogens in the whole community. - The genes do not support a higher loading rate before the next sample. (Schnürer & Jarvis 2018)
Why: The acetate route is thin: Methanosarcina flavescens, 0.14 % 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. At T1 no genome carried the acetate route. - This assay does not measure trace elements (Fe, Co, Ni, Se, Mo, W). In plants on energy crops a lack is common. 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: 4.2 %. Acetate route: Methanosarcina flavescens, 0.14 % of mapped reads. Studies link a lack of selenium and cobalt to propionate accumulation. The H₂/CO₂ route was 7.9 % at T1. - If acetate accumulates, digestate from a reactor with similar feed and temperature and an established acetate route is one option, after a lower load and a search for the cause. Source and animal by-product rules are the operator's call. (FNR 2010; Schnürer & Jarvis 2018)
Why: There is no known acetate-using methanogen above the reporting cutoff of this report (0.1 % of reads), or only one at a very low share: Methanosarcina flavescens, 0.14 % of mapped reads. A route below the cutoff, or one too thin to assemble at this sequencing depth, is not shown here.
Samples
| T1 | T2 | |
|---|---|---|
| Feed | 72 % maize silage, 28 % pig manure | 67 % maize silage, 33 % pig manure |
| Retention time (average days the material stays in the reactor) | 55 d | 81 d |
| Feed NH₄⁺-N | 1.06 g/kg | 1.25 g/kg |
| Feed acetate · total VFA | 3.4 · 4.2 g/kg | 4.0 · 4.9 g/kg |
| Genomes scored (share of reads) | 105 (90.6 %) | 115 (89.5 %) |
The two samples come from two different studies. Part of the change may come from different DNA extraction and sequencing. A client series is sampled and processed the same way every time.
Methanogens
| Genome | Route | T1 | T2 | Change |
|---|---|---|---|---|
| Methanoculleus thermohydrogenotrophicum | H₂/CO₂ | 3.80 % | 0.60 % | decreased |
| Methanoculleus sp. | H₂/CO₂ | 2.92 % | 1.22 % | decreased |
| Methanoculleus bourgensis | H₂/CO₂ | 0.79 % | 1.63 % | below threshold |
| Methanosarcina flavescens | acetate · H₂/CO₂ · methyl | — | 0.1 % | new |
| 3 other H₂/CO₂ methanogens | H₂/CO₂ | < 0.5 % | < 0.5 % | below floor |
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.
Genomes that changed
30 of 59 genomes above 0.5 % changed. The largest are shown. The full list is in genomes_over_time.tsv.
Increased or new at T2
Decreased or lost at T2
The new genomes break down proteins and amino acids (Cloacimonadota, Fusobacteriota, Saccharofermentanales). This matches the higher ammonium in the feed.
Breakdown capacity
| Capacity | T1 genomes | share | T2 genomes | share | Change |
|---|---|---|---|---|---|
| Cellulose and hemicellulose | 22 | 14.1 % | 18 | 8.2 % | decreased |
| of which anchored (built to work outside the cell) | 8 | 3.2 % | 9 | 2.9 % | no change |
| Lipids | 7 | 2.3 % | 11 | 3.9 % | increased |
| Proteins | 71 | 41.4 % | 71 | 40.4 % | no change |
| Propionate pathway | 19 | 11.0 % | 14 | 12.0 % | no change |
| Ammonia release (ethanolamine) | 2 | 0.84 % | 1 | 0.12 % | below threshold |
On maize silage, cellulose breakdown sets the rate. If gas yield per tonne of silage has decreased too, consider a longer retention time or feed pretreatment.
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".
| 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. |
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. Change rule: at least 1 percentage point and at least 1.5-fold. Genome floor: 0.5 %. The scripts in the digestome repository make every number again from the public inputs. Literature behind the suggestions. 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.