Thermophilic and mesophilic sulfate reduction by rare biosphere bacteria in acidic metal-bearing mine wastes from the temperate climate zone.
The main results reproduced, with only marginal, non-material deviations.
Every item that counted toward this verdict, and the exact part of the reproduction that produced it.
- Nothing in this column.
- 🟡Could not use the authors’ exact input data
- 🟡Reported values were only indirectly comparable
- 🟡A deviation arose in the data or preprocessing
- 🔴A deviation was attributed to the published material
- 🟡Reported values were not (fully) derivable from the shared data
- 🟡The deviation was non-trivial in magnitude
- 🟡The central claim did not (fully) hold under reproduction
- 🟡Overall, the reproduction showed a material discrepancy
A 0–100 reproducibility-quality score from the per-question grades, shown as a z-score: standard deviations above (+) or below (−) the mean of comparable assessments.
▸Reproduction agent’s raw note
Third-party-tool reproduction (Sickle + the paper's described amplicon pipeline) of a paper with no authors' repo. ONE clean EXACT data point: the Bu28s metagenome read-pair count 258,978,449 equals the authoritative SRA deposited spot count byte-for-byte (confirmed from archive, not a wasteful 32GB recount). The 16S community-profiling percentages are only PARTIALLY reproduced: re-running Cutadapt->FLASH->Sickle->VSEARCH-OTU97->SINTAX/SILVA on the paper's own amplicon reads recovers the same SET of dominant acidophile genera for the anchored Bu28 sediment sample (Sulfobacillus close at 25.9 vs 22.1% of assigned reads; Metallibacterium/Leptospirillum/Acidithiobacillus present but ~1.5-2x low), but it is NOT a 1:1 match - ranks differ, Ferrimicrobium is ~4x over-represented, and >60% of reads are genus-unassigned. The Methods do not specify enough (full-SILVA-v132 SINTAX reference vs the only-obtainable DADA2 trainset, proprietary Usearch vs VSEARCH, SINTAX cutoff, chimera/singleton/normalization details) to reproduce the exact percentages - this is the hard ~20%. NOTABLE FLAG for human review (not asserted as fabrication): the paper's #1 Bu29-mat taxon Sulfacidibacillus (32.0%) is a genus described in 2019, absent from SILVA v132 (Dec 2017) which the Methods cite - a v132 pipeline cannot output it, so either a newer/curated reference was actually used or the assignment came from elsewhere. NOT attempted: DsrAB metagenomic read counts (heavy RPS-BLAST/Diamond over 259M reads) and all wet-lab results (cultivation, 35S rates, isolate genomes).
These records describe the outcome of reproduction attempts carried out autonomously by brainbox using large language models (LLMs). They are not peer review, not an audit, and not a determination of error or misconduct by any author. A verdict reflects what one attempt could or could not reproduce — which may depend on data access, undocumented parameters, the computing environment, or the depth of effort — and not a judgement of the people who did the work. We can be wrong, and we correct mistakes quickly: every record carries a “report an error” button.
Assessment versions
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v1 current initial assessment Score 52assessed: 2026-06-16 ⛓ 66470f32b912
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Provenance — full disclosure
When this reproduction was carried out, which methodology version was used, and by whom — so the record can be audited and checked independently.
- Reproduced
- 2026-06-16
- Rubric version
- v1.0
- Assessed by
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🤖 AI curator · claude (ai-curator room) · v1.0 · run #1 2026-06-16no human curator yet
- Last updated
- 2026-08-05
Provisional, curator- or AI-assessed, and independently checkable. A reproduction outcome states what one attempt could reproduce — not a judgement of the authors.
Deep full-text extraction
Model: opusActive dissimilatory sulfate reduction occurs in temperate acidic metalliferous mine tailings, and thermophilic sulfate-reducing bacteria may be present and active even at this mesothermic AMD site, with low-abundance 'rare biosphere' SRB driving the geochemically important process.
- ★ High in-situ sulfate reduction rates occur in acidic Bom-Gorkhon tailings sediments (up to 9.86 µmol SO4 cm-3 day-1 at 20 °C) finding
- ★ Thermophilic sulfate reduction of comparable magnitude occurs at 60 °C in these temperate tailings, implying an active thermophilic SRB community finding
- ★ Geochemically important active sulfate reduction is carried out by a low-abundance 'rare biosphere' consortium, with Desulfosporosinus and other SRB at low abundance finding
- ★ Metagenomic dsrAB mapping assigned most dissimilatory sulfate-reducer reads to uncultured Acidobacteriota, with Desulfosporosinus a minority finding
- ★ Thermophilic spore-forming Desulfotomaculum and Desulfofundulus are likely responsible for the thermophilic process, with Desulfofundulus thermospores potentially germinating at 20 °C mechanism
- ★ Strains BG-T and OT are described as a new species, Desulfosporosinus cupriresistens sp. nov. resource
- Strain BG-T is acidophilic (pH 1.3–5.5, optimum 4.0), copper-tolerant (up to 8 g Cu2+ L-1), and uses diverse electron donors for sulfate reduction finding
- SRR was measured with 35S-sulfate radioactive tracer and community composition by 16S rRNA gene profiling plus metagenomics method
| Assay | System | Perturbation | Readout | Platform |
|---|---|---|---|---|
| radioactive 35SO4 2- tracer sulfate-reduction-rate measurement | acidic mine tailing sediments, water and microbial mats (Bom-Gorkhon, Transbaikalia) | incubation temperature (20 °C in situ vs 60 °C) | sulfate reduction rate as reduced sulfur in AVS and CRS fractions (µmol SO4 cm-3 day-1) | — |
| 16S rRNA gene amplicon profiling | water and sediment samples Bu27w, Bu27s, Bu28w, Bu28s, Bu29 mat | none | relative abundance of taxonomic groups (genus level) | — |
| shotgun metagenomic sequencing with dsrA/dsrB gene mapping | sediment sample Bu28s | none | reads mapped to dsrA/dsrB and taxonomic assignment of sulfate-reducer lineages | — |
| physicochemical/geochemical characterization (pH, Eh, sulfate, dissolved metals) | tailings leachate water and sediments | none | pH, Eh, SO4, Fe, Al, Zn, Cu, Cd concentrations | — |
| X-ray diffraction mineralogy | tailings sediments Bu27, Bu28 | none | mineral phase identification (clinochlore, muscovite, gypsum, hotsonite) | — |
| transmission electron microscopy (TEM) of ultrathin sections | Desulfosporosinus strain BG-T and OT cultures | copper addition (100 mg Cu2+ L-1, 1.57 mM) vs no metal | cell ultrastructure, EPS layer, vacuole-like globules, spores | — |
| physiological growth/cultivation characterization | Desulfosporosinus strains BG-T and OT | pH, temperature, NaCl, electron donors/acceptors, Cu2+ concentration | growth ranges, substrate utilization, copper tolerance, fatty acid composition | — |
| phylogenomic analysis (ANI/AAI, 120 marker gene tree) | genomes of strains BG-T and OT and relatives | none | average nucleotide/amino acid identity and tree topology for species delineation | — |
- ▲ In-situ SRR in puddle sediment Bu27s was high at 20 °C 9.86 ± 0.89 µmol SO4 cm-3 day-1
- ▲ SRR at 60 °C was the same order of magnitude as in-situ, peaking in Bu29 mat 14.4 ± 0.681 µmol SO4 cm-3 day-1
- – AVS was the major product of 35SO4 reduction 91–100%
- ▼ Desulfosporosinus phylotype present at minor abundance in Bu28 water/sediment 0.01% (water), 0.13% (sediment)
- ▼ Relative abundance of sulfate reducers in Bu28s metagenome estimated very low ~0.07% of microbial population
- – Most dsrAB reads assigned to uncultured Acidobacteriota; ~quarter to Desulfosporosinus 78 of 144 reads Acidobacteriota
- – Strain OT shows high ANI with BG-T, near species threshold ANI 94.28%
- – Closest uncultivated relative Ca. Desulfosporosinus infrequens distinct from BG-T ANI 81.37% / AAI 78.07%
- other 9.86 ± 0.89 µmol SO4 cm-3 day-1 (max in-situ (20 °C) SRR, sediment Bu27s)
- other 14.4 ± 0.681 µmol SO4 cm-3 day-1 (max SRR at 60 °C, mat Bu29)
- other 0.22 ± 0.287 µmol SO4 cm-3 day-1 (lowest in-situ SRR, sediment Bu28s)
- count 489 and 1322 reads mapped to dsrA and dsrB (of ~518 million metagenome reads from Bu28s)
- count 144 reads assigned to dissimilatory sulfate-reducing lineages; 78 to Acidobacteriota (dsrAB taxonomic assignment)
- fold_change ANI 94.28% (OT vs BG-T genome identity)
- other pH 2.37–2.55; SO4 up to 3,676 mg L-1; Fe up to 471 mg L-1 (water sample geochemistry)
- other C16:1 ω7 56.3%, C16:0 26.6% (major cellular fatty acids of strain BG-T)
Statistical methods review
Model: sonnetA neutral, descriptive read of the statistical approach — what was done, and (for shared learning, not as criticism) what could also have been done.
This environmental microbiology study quantifies sulfate reduction rates (SRR) in acidic mine tailings using radioactive 35S-sulfate tracer incubations reported as mean ± SD, with parallel incubations at 20 °C and 60 °C. Microbial community structure is characterised descriptively via 16S rRNA relative-abundance profiling and dsr gene read-mapping from metagenomics. Novel isolate species status is assessed through pairwise average nucleotide identity (ANI) and average amino acid identity (AAI) against a 95% threshold, supported by multi-method phylogenetic tree reconstruction.
| Test | Applied to | n | Assumptions |
|---|---|---|---|
| Radioactive 35SO4 tracer quantification; results expressed as mean ± SD | SRR in sediment and water samples at 20 °C and 60 °C across sites Bu27s, Bu27w, Bu28s, Bu28w, Bu29 (Fig. 2) | 'Parallel syringes' mentioned; exact replicate count not stated | not stated |
| Average nucleotide identity (ANI) and average amino acid identity (AAI) against 95 % species threshold | Species delineation of strains BGT and OT vs. known Desulfosporosinus spp. (Fig. 6 and text) | Pairwise genome comparisons; number of reference genomes not stated | not stated |
| Neighbour-joining phylogenetic tree construction, corroborated by maximum-likelihood and minimum-evolution trees | Phylogenetic placement using concatenated 120 bacterial single-copy marker proteins (Fig. 6) | null | not stated |
| 16S rRNA amplicon relative-abundance profiling (read-proportion percentages) | Microbial community composition across all five samples (Fig. 3) | null | not stated |
| Metagenomic short-read mapping to dsrA/dsrB reference databases; relative abundance estimated from mapped read proportions assuming average gene (~1 kb) and genome (~5 Mbp) size | Sulfate-reducer abundance estimation in Bu28s metagenome (Fig. 4); ~518 million total reads, 144 assigned to sulfate-reducing lineages | Single metagenome from one sample (Bu28s) | not stated |
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SRR at 20 °C and 60 °C were compared descriptively across sites without a formal statistical test↳ Could also: A paired or unpaired t-test, Mann-Whitney U test, or a mixed-effects model could be applied to test whether SRR differs between temperature conditions — A formal test would quantify the probability that the observed temperature similarity reflects a true biological pattern rather than measurement variability, which is particularly informative given that the authors themselves describe the 60 °C result as unexpected
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SRR dispersion is reported as SD with an unstated number of replicate syringes↳ Could also: Reporting 95 % confidence intervals together with an explicit replicate count (n) would also convey precision — CIs communicate directly how precisely the mean is estimated and facilitate cross-study comparison; explicit n allows readers to distinguish SD from SEM and to assess whether the spread reflects biological variability or measurement uncertainty
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Microbial community composition was characterised using relative abundance percentages from 16S rRNA profiling across five samples↳ Could also: Alpha-diversity indices (e.g., Shannon, Chao1) and beta-diversity ordination (e.g., Bray-Curtis dissimilarity with PERMANOVA) could also be computed — Ordination and permutation-based tests provide a structured framework for testing whether community structure differs significantly between water and sediment habitats or between sites, complementing descriptive percentage summaries
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Species delineation relied on ANI and AAI point estimates against a 95 % threshold↳ Could also: Digital DNA-DNA hybridization (dDDH, e.g., via TYGS/GGDC) could be applied as a complementary genomic species-boundary method — dDDH is an independently validated and widely cited prokaryotic species standard; reporting it alongside ANI/AAI strengthens novel species descriptions by satisfying multiple community conventions simultaneously
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Metagenomic sulfate-reducer relative abundance was estimated from mapped dsr read counts using assumed average gene and genome sizes↳ Could also: Genome-resolved metagenomics (metagenome-assembled genomes, MAGs) with coverage-based abundance estimation could also be applied — MAG-based estimates avoid reliance on assumed gene and genome lengths and enable direct linkage of dsr metabolic potential to a taxonomically resolved genomic bin, potentially improving both accuracy and taxonomic resolution
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Phylogenetic placement used a neighbour-joining tree as the primary method, with ML and ME as verification↳ Could also: Bayesian inference (e.g., MrBayes, PhyloBayes) producing posterior probability support values could also serve as the primary reconstruction method — Bayesian methods express branch support as posterior probabilities and can incorporate richer substitution models; they are increasingly standard in phylogenomics and would provide complementary confidence metrics to bootstrap values
Citation network
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What was reproduced
The exact results taken into scope, with each reported value next to the value our attempt produced.
Scope — pmid-41345487
Paper: Karnachuk et al. 2025, Sci Rep — "Thermophilic and mesophilic sulfate reduction by rare biosphere bacteria in acidic metal-bearing mine wastes from the temperate climate zone." DOI 10.1038/s41598-025-28271-4.
Data: SRA BioProject PRJNA1244608
- 16S rRNA amplicon (V3–V6, primers 341F/806R, Illumina MiSeq 2×300): 5 runs SRR32944694, SRR32944695, SRR32944696, SRR32944697, SRR32944698 (experiments SRX28218996–SRX28219000). Sample SAMN47710685 ("sediment metagenome", run SRR32944695) = Bu28 sediment.
- Metagenome (WGS, Illumina HiSeq2500 2×150) of sample Bu28s: run SRR32963565 (SRX28237238), read_count 258,978,449 pairs per ENA.
Code (per Data/Code availability + Methods): No authors' own repository. The named code artifact is the third-party trimming tool Sickle (github.com/najoshi/sickle). Per BRIEF P16, applying the described third-party pipeline to the paper's own data is an equally valid reproduction. Full amplicon pipeline named in Methods: Cutadapt v4.0 → Sickle v1.33 → FLASH v1.2.11 (merge) → chimera+singleton removal → Usearch (OTU clustering, 97% id) → VSEARCH sintax vs SILVA v132.
In scope (pipeline-derived; attempted)
| ID | Reported result | Pipeline | Plan |
|---|---|---|---|
| C1 | Metagenome: 258,978,449 read pairs (Bu28s) | sequencing/deposit | Confirm vs deposited SRA spot count; optional real recount on «our HPC» |
| C2 | Bu28 sediment 16S dominant taxa: Metallibacterium 23.3%, Sulfobacillus 22.1%, Leptospirillum 15.6% | Cutadapt→Sickle→FLASH→OTU97→sintax/SILVA | Run pipeline on SRR32944695, compute genus rel. abundance |
| C3 | Bu29 mat 16S dominant taxa: Sulfacidibacillus 32.0%, Leptospirillum 17.4%, Acidiphilium 12.7% | same | Run pipeline on all amplicon runs, identify Bu29 by profile, compare |
Out of scope (not attempted — the hard ~20% / wet-lab / external)
- DsrAB read counts (DsrA 489, DsrB 1322; Acidobacteriota 78; Desulfosporosinus ~36): requires RPS-BLAST v2.9.0 vs CDD (TIGR02064/02066) + Diamond vs NCBI-NR over 259M-read metagenome — heavy compute, fuzzy thresholds, low headline value. Deferred per 80/20; will attempt only if amplicon reproduction leaves budget.
- Wet-lab: cultivation, sulfate-reduction rates (³⁵S radiotracer), isolate physiology, GenBank isolate 16S (PV524097 etc.) — manual/experimental, not a pipeline.
Deviations recorded up front
- Usearch → VSEARCH for OTU clustering: Usearch 64-bit is proprietary;
VSEARCH
--cluster_sizeat 0.97 is the standard open equivalent (the paper already used VSEARCH for the sintax step). Noted as a method deviation. - SILVA v132 → v138.1 SINTAX reference if v132 sintax-formatted DB is not readily fetchable; dominant acidophile genera here are robust to DB version. Actual version used recorded in environment.lock.
Assessments & scoring basis
Each contributor’s verdict, the per-question basis, and the auditable, itemised worksheet behind it.
An automated assessment. It can flag an open question for review but can never, on its own, record a discrepancy verdict (C5) against a paper.
Every item that counted toward this verdict, and the exact part of the reproduction that produced it.
C1 reproduces exactly (258,978,449 read pairs equals the authoritative SRA deposited spot count) and the Bu28 sediment dominant-genus set is qualitatively recovered, with Sulfobacillus close (25.9 vs 22.1% of assigned reads). The percentage mismatches are mostly on our side / underspecification: open v138.1 trainset vs full SILVA v132, VSEARCH vs Usearch, unstated cutoffs, and a different denominator (>60% reads genus-unassigned), producing ~1.5-2x shifts and a ~4x Ferrimicrobium over-representation. The one authors-side flag is real but not fabrication-grade: the Bu29 headline taxon Sulfacidibacillus (32.0%) postdates the cited SILVA v132 and cannot come from the stated pipeline, so a reviewer should establish which reference actually produced it. Overall a solid partial reproduction with explainable deviations — yellow, not critical.
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Reproduction footprint
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