# MIX-MB(M) — Microbes

## Microbial Component Standards (M)

This document identifies Minimum Information (MI) required to report microbial organisms involved in biotransformation of xenobiotics, ensuring comprehensive documentation of bacterial, and archaeal species/strains used in biotransformation studies and information on the assays.

**Author:** Mahnoor Zulfiqar

**Part of:** MIX-MB Standard\_main\
**Compatible with:**

* MIX-MB(X)
* MIX-MB(B) **Alignment:** NCBI Taxonomy, MIxS, ChEMBL, FAIR principles

***

***

## 1. Overview

MIX-MB(M) establishes minimum information standards for reporting microbial organisms in xenobiotic biotransformation studies, ensuring data is:

* **Findable:** Properly annotated with taxonomic identifiers and strain information
* **Accessible:** Standardized formats compatible with public databases
* **Interoperable:** Uses community ontologies and controlled vocabularies
* **Reusable:** Complete provenance, cultivation, and experimental conditions

### 1.4 Identifiers and Cross-Referencing

**This is the first practical step: name your organisms properly before filling in any other field.**

#### Organism Identifiers

Each microbial organism in a MIX-MB submission must be identified with an **NCBI TaxID** — the primary organism identifier used across all MIX-MB files. This value populates `ASSAY_TAX_ID` in `ASSAY.tsv`.

**Identifier priority order:**

| Priority | Identifier                    | Field                                       | Example                 |
| -------- | ----------------------------- | ------------------------------------------- | ----------------------- |
| 1        | **NCBI TaxID**                | `identifier` / `ASSAY_TAX_ID`               | `1351`                  |
| 2        | **LPSN scientific name**      | `name` / `ASSAY_ORGANISM`                   | `Enterococcus faecalis` |
| 3        | **Culture collection ID**     | `additionalProperty` → `culture_collection` | `ATCC 19433`            |
| 4        | **Genome assembly accession** | `additionalProperty` → `genome_assembly`    | `GCA_000007785.1`       |

NCBI TaxID is **mandatory** for all organisms. If a strain does not yet have a registered TaxID (e.g. a novel isolate), report at the lowest available taxonomic rank and note the absence of a strain-level TaxID in `ACTIVITY_COMMENT`.

#### Assay Index (AIDX)

Each assay — representing one organism × condition combination × type — is assigned an **AIDX**, which links `ASSAY.tsv`, `ASSAY_PARAM.tsv`, and `ACTIVITY.tsv`.

**Minting rules:**

* Format: `[FirstAuthorLastName]_[Genus]_[species]_[ConditionOrNote]_[type]`, e.g. `Zimmermann_Actinomyces_graevenitzii_anaerobic_biotransformation`
* AIDXs must be unique within a study
* Use only ASCII letters, digits, underscores, and hyphens — no spaces
* add protein ID, if its an enzyme assay
* Each distinct organism–condition combination gets its own AIDX; do not reuse AIDXs across different strain batches or oxygen conditions

#### Minting Scheme for Uncharacterised or Novel Organisms

| Situation                          | AIDX format                                    | `ASSAY_ORGANISM` value      | `ASSAY_TAX_ID`                                |
| ---------------------------------- | ---------------------------------------------- | --------------------------- | --------------------------------------------- |
| Known species, known strain        | `[Author]_[Genus]_[species]_[Strain]`          | Full binomial name          | Registered species TaxID                      |
| Known species, undesignated strain | `[Author]_[Genus]_[species]_unknown_strain`    | Full binomial name          | Species-level TaxID                           |
| Novel isolate (unclassified)       | `[Author]_unclassified_[IsolateID]`            | `unclassified bacteria`     | `2` (root Bacteria) or closest assigned TaxID |
| Mixed / community assay            | `[Author]_mixed_community_[SourceDescription]` | `mixed microbial community` | `1` (root) or metagenome TaxID                |

***

## 2. Bioschemas

MIX-MB(M) integrates established Bioschemas profiles for structured, FAIR-compliant annotation of microbial organisms, strains, and their biotransformation-relevant molecular components. Bioschemas fulfils the Findability and Interoperability aspects of FAIR.

The following profiles are used:

| Profile                                                                          | Bioschemas Group | Version      | Use in MIX-MB(M)                          |
| -------------------------------------------------------------------------------- | ---------------- | ------------ | ----------------------------------------- |
| [Taxon](https://bioschemas.org/profiles/Taxon/1.0-RELEASE)                       | Biodiversity     | 1.0-RELEASE  | Organism taxonomy identification          |
| [TaxonName](https://bioschemas.org/profiles/TaxonName/1.0-RELEASE)               | Biodiversity     | 1.0-RELEASE  | Structured scientific name representation |
| [Sample](https://bioschemas.org/profiles/Sample/0.2-RELEASE-2018_11_10)          | Samples          | 0.2-RELEASE  | Microbial culture sample metadata         |
| [Gene](https://bioschemas.org/profiles/Gene/1.0-RELEASE)                         | Genes            | 1.0-RELEASE  | Biotransformation-related genes           |
| [Protein](https://bioschemas.org/profiles/Protein/0.11-RELEASE)                  | Proteins         | 0.11-RELEASE | Biotransformation enzymes                 |
| [ProteinAnnotation](https://bioschemas.org/profiles/ProteinAnnotation/0.6-DRAFT) | Proteins         | 0.6-DRAFT    | Enzyme functional annotations             |

***NOTE:*** Taxon, TaxonName, and Sample profiles map to the `ASSAY.tsv` and `ASSAY_PARAM.tsv` ChEMBL submission files. Gene, Protein, and ProteinAnnotation profiles are for extended metadata (Section 6.2) and are optional but strongly recommended for Gold-standard submissions.

***

### 2.1 Taxon Profile

Use [Bioschemas Taxon 1.0-RELEASE](https://bioschemas.org/profiles/Taxon/1.0-RELEASE) to identify and classify the microbial organism involved in the biotransformation study.

**Minimum Properties:**

| Property         | Expected Type | Cardinality | Description                                           |
| ---------------- | ------------- | ----------- | ----------------------------------------------------- |
| `@context`       | URL           | ONE         | `"https://schema.org/"`                               |
| `@type`          | Text          | ONE         | `"Taxon"`                                             |
| `@id`            | IRI           | ONE         | Globally unique IRI (e.g., NCBI Taxonomy URL)         |
| `dct:conformsTo` | IRI           | ONE         | `"https://bioschemas.org/profiles/Taxon/1.0-RELEASE"` |
| `name`           | Text          | ONE         | Currently valid scientific name for the taxon         |

**Recommended Properties:**

| Property         | Expected Type            | Cardinality | Description                                                    |
| ---------------- | ------------------------ | ----------- | -------------------------------------------------------------- |
| `taxonRank`      | PropertyValue, Text, URL | MANY        | Taxonomic rank, preferably as URI from a controlled vocabulary |
| `parentTaxon`    | Taxon, Text, URL         | ONE         | Closest parent taxon                                           |
| `sameAs`         | URL                      | MANY        | Reference URLs identifying the taxon (NCBI, Wikidata, etc.)    |
| `scientificName` | TaxonName                | ONE         | Structured TaxonName object (see Section 2.2)                  |

**Optional Properties (MIX-MB(M) relevant):**

| Property                  | Expected Type            | Cardinality | Description                                      |
| ------------------------- | ------------------------ | ----------- | ------------------------------------------------ |
| `identifier`              | PropertyValue, Text, URL | ONE         | Authority identifier (e.g., NCBI TaxID, GBIF)    |
| `alternateName`           | Text                     | MANY        | Synonyms or former names                         |
| `alternateScientificName` | TaxonName                | MANY        | Synonym scientific names                         |
| `childTaxon`              | Taxon, Text, URL         | MANY        | Known subspecies or strains                      |
| `dwc:vernacularName`      | Text                     | MANY        | Common name (e.g., "E. coli")                    |
| `url`                     | URL                      | ONE         | Link to the taxon record in a reference database |
| `description`             | Text                     | ONE         | Free-text description of the taxon               |

***NOTE:*** `name` and `identifier` from this profile map directly to `ASSAY_ORGANISM` and `ASSAY_TAX_ID` in `ASSAY.tsv`.

***

### 2.2 TaxonName Profile

Use [Bioschemas TaxonName 1.0-RELEASE](https://bioschemas.org/profiles/TaxonName/1.0-RELEASE) as a structured representation of the organism's scientific name — used as the value of `scientificName` in Section 2.1.

**Minimum Properties:**

| Property         | Expected Type | Cardinality | Description                                               |
| ---------------- | ------------- | ----------- | --------------------------------------------------------- |
| `@context`       | URL           | ONE         | `"https://schema.org/"`                                   |
| `@type`          | Text          | ONE         | `"TaxonName"`                                             |
| `@id`            | IRI           | ONE         | Globally unique IRI (e.g., LPSN URL)                      |
| `dct:conformsTo` | IRI           | ONE         | `"https://bioschemas.org/profiles/TaxonName/1.0-RELEASE"` |

**Recommended Properties:**

| Property    | Expected Type            | Cardinality | Description                                                  |
| ----------- | ------------------------ | ----------- | ------------------------------------------------------------ |
| `name`      | Text                     | ONE         | Taxon name without authorship or date information            |
| `taxonRank` | PropertyValue, Text, URL | MANY        | Taxonomic rank, preferably as URI                            |
| `author`    | Person, Organization     | MANY        | Authorship and date details per nomenclatural rules          |
| `sameAs`    | URL                      | MANY        | URLs to additional name records (LPSN, Index Fungorum, etc.) |
| `url`       | URL                      | ONE         | Webpage for this taxon name record                           |

**Optional Properties:**

| Property      | Expected Type              | Cardinality | Description                                      |
| ------------- | -------------------------- | ----------- | ------------------------------------------------ |
| `identifier`  | PropertyValue, Text, URL   | ONE         | Authority identifiers (GBIF, WoRMS, etc.)        |
| `isBasedOn`   | CreativeWork, Product, URL | MANY        | Publication or resource underlying this name     |
| `description` | Text                       | ONE         | Description of the name or nomenclatural context |

***

### 2.3 Sample Profile

Use [Bioschemas Sample 0.2-RELEASE](https://bioschemas.org/profiles/Sample/0.2-RELEASE-2018_11_10) for individual microbial culture samples used in biotransformation assays. Each sample represents a specific strain in a specific cultivation condition.

**Minimum Properties:**

| Property         | Expected Type            | Cardinality | Description                                                       |
| ---------------- | ------------------------ | ----------- | ----------------------------------------------------------------- |
| `@context`       | URL                      | ONE         | `"https://schema.org/"`                                           |
| `@type`          | Text                     | ONE         | `"Sample"`                                                        |
| `@id`            | IRI                      | ONE         | Globally unique IRI (e.g., NCBI BioSample URL)                    |
| `dct:conformsTo` | IRI                      | ONE         | `"https://bioschemas.org/profiles/Sample/0.2-RELEASE-2018_11_10"` |
| `identifier`     | PropertyValue, Text, URL | MANY        | Unique sample identifier (e.g., `biosample:SAMN12345`, AIDX)      |

**Recommended Properties:**

| Property | Expected Type | Cardinality | Description                                       |
| -------- | ------------- | ----------- | ------------------------------------------------- |
| `url`    | URL           | ONE         | URL of the sample record in a database or biobank |

**Optional Properties (MIX-MB(M) relevant — use `additionalProperty`):**

| `additionalProperty` name | Value   | Description                                                         |
| ------------------------- | ------- | ------------------------------------------------------------------- |
| `strain`                  | Text    | Strain designation (e.g., K-12 substr. MG1655, ATCC 25922)          |
| `culture_collection`      | Text    | Culture collection identifier (e.g., ATCC, DSMZ, CGSC)              |
| `oxygen_requirement`      | CV term | Oxygen condition (see Section 7.1 for controlled vocabulary)        |
| `growth_medium`           | Text    | Medium name (e.g., BHI, M9 minimal medium, LB broth)                |
| `incubation_temperature`  | Text    | Growth temperature with unit (e.g., "37°C")                         |
| `sample_type`             | CV term | Sample type (see Section 7.3 for controlled vocabulary)             |
| `harvest_phase`           | CV term | Growth phase at harvest (see Section 7.4 for controlled vocabulary) |
| `biosafety_level`         | Text    | BSL classification (e.g., "BSL-1", "BSL-2")                         |

***NOTE:*** This profile maps to `ASSAY.tsv` (strain, cell type, assay organism fields) and `ASSAY_PARAM.tsv` (growth conditions) for ChEMBL submission.

***

### 2.4 Gene Profile

Use [Bioschemas Gene 1.0-RELEASE](https://bioschemas.org/profiles/Gene/1.0-RELEASE) to annotate biotransformation-relevant genes in the microbial genome (e.g., genes encoding nitroreductases, azoreductases, dehalogenases, or other xenobiotic-metabolising enzymes).

**Minimum Properties:**

| Property         | Expected Type            | Cardinality | Description                                          |
| ---------------- | ------------------------ | ----------- | ---------------------------------------------------- |
| `@context`       | URL                      | ONE         | `"https://schema.org/"`                              |
| `@type`          | Text                     | ONE         | `"Gene"`                                             |
| `@id`            | IRI                      | ONE         | Globally unique IRI (e.g., NCBI Gene URL)            |
| `dct:conformsTo` | IRI                      | ONE         | `"https://bioschemas.org/profiles/Gene/1.0-RELEASE"` |
| `identifier`     | PropertyValue, Text, URL | ONE         | Gene identifier (e.g., NCBI Gene ID, locus tag)      |
| `name`           | Text                     | ONE         | Gene symbol or name (e.g., "nfsA", "porA")           |

**Recommended Properties:**

| Property                | Expected Type | Cardinality | Description                                        |
| ----------------------- | ------------- | ----------- | -------------------------------------------------- |
| `description`           | Text          | ONE         | Gene function description                          |
| `encodesBioChemEntity`  | BioChemEntity | MANY        | Protein(s) or RNA encoded by this gene             |
| `isPartOfBioChemEntity` | BioChemEntity | MANY        | Chromosome or genome this gene belongs to          |
| `url`                   | URL           | ONE         | Link to the gene record in NCBI Gene or equivalent |

**Optional Properties (MIX-MB(M) relevant):**

| Property                        | Expected Type                   | Cardinality | Description                                                     |
| ------------------------------- | ------------------------------- | ----------- | --------------------------------------------------------------- |
| `hasMolecularFunction`          | DefinedTerm, PropertyValue, URL | MANY        | GO molecular function term                                      |
| `isInvolvedInBiologicalProcess` | DefinedTerm, PropertyValue, URL | MANY        | GO biological process term                                      |
| `taxonomicRange`                | DefinedTerm, Taxon, Text, URL   | MANY        | Organism(s) expressing this gene                                |
| `hasBioPolymerSequence`         | Text                            | ONE         | Nucleotide sequence (FASTA)                                     |
| `alternateName`                 | Text                            | MANY        | Alternative gene names or synonyms                              |
| `sameAs`                        | URL                             | MANY        | Same gene in other databases (UniProt gene page, Ensembl, etc.) |

***

### 2.5 Protein Profile

Use [Bioschemas Protein 0.11-RELEASE](https://bioschemas.org/profiles/Protein/0.11-RELEASE) to describe enzymes responsible for xenobiotic biotransformation encoded by the microorganism.

**Minimum Properties:**

| Property         | Expected Type            | Cardinality | Description                                              |
| ---------------- | ------------------------ | ----------- | -------------------------------------------------------- |
| `@context`       | URL                      | ONE         | `"https://schema.org/"`                                  |
| `@type`          | Text                     | ONE         | `"Protein"`                                              |
| `@id`            | IRI                      | ONE         | Globally unique IRI (e.g., UniProt URL)                  |
| `dct:conformsTo` | IRI                      | ONE         | `"https://bioschemas.org/profiles/Protein/0.11-RELEASE"` |
| `identifier`     | PropertyValue, Text, URL | ONE         | Protein identifier (e.g., UniProt accession, PDB ID)     |
| `name`           | Text                     | ONE         | Protein name                                             |

**Recommended Properties:**

| Property                   | Expected Type                 | Cardinality | Description                                     |
| -------------------------- | ----------------------------- | ----------- | ----------------------------------------------- |
| `description`              | Text                          | ONE         | Protein function; start with "Function: \[...]" |
| `isEncodedByBioChemEntity` | Gene                          | MANY        | Gene(s) encoding this protein                   |
| `taxonomicRange`           | DefinedTerm, Taxon, Text, URL | MANY        | Organism(s) expressing this protein             |
| `url`                      | URL                           | ONE         | Link to the protein record (UniProt, etc.)      |

**Optional Properties (MIX-MB(M) relevant):**

| Property                         | Expected Type                   | Cardinality | Description                                         |
| -------------------------------- | ------------------------------- | ----------- | --------------------------------------------------- |
| `hasMolecularFunction`           | DefinedTerm, PropertyValue, URL | MANY        | GO molecular function term                          |
| `isInvolvedInBiologicalProcess`  | DefinedTerm, PropertyValue, URL | MANY        | GO biological process term                          |
| `isLocatedInSubcellularLocation` | DefinedTerm, PropertyValue, URL | MANY        | Subcellular localisation                            |
| `bioChemInteraction`             | BioChemEntity                   | MANY        | Interacting molecules (substrates, cofactors)       |
| `hasBioPolymerSequence`          | Text                            | ONE         | Amino acid sequence (FASTA)                         |
| `alternateName`                  | Text                            | MANY        | Protein synonyms                                    |
| `sameAs`                         | URL                             | MANY        | Same protein in other databases (PDB, STRING, etc.) |

***

### 2.6 ProteinAnnotation Profile

Use [Bioschemas ProteinAnnotation 0.6-DRAFT](https://bioschemas.org/profiles/ProteinAnnotation/0.6-DRAFT) to annotate specific functional features of biotransformation enzymes — such as active sites, binding sites, EC number assignments, or domain annotations.

**Minimum Properties:**

| Property         | Expected Type            | Cardinality | Description                                                     |
| ---------------- | ------------------------ | ----------- | --------------------------------------------------------------- |
| `@context`       | URL                      | ONE         | `"https://schema.org/"`                                         |
| `@type`          | Text                     | ONE         | `"ProteinAnnotation"`                                           |
| `@id`            | IRI                      | ONE         | Globally unique IRI (e.g., UniProt feature URL)                 |
| `dct:conformsTo` | IRI                      | ONE         | `"https://bioschemas.org/profiles/ProteinAnnotation/0.6-DRAFT"` |
| `identifier`     | PropertyValue, Text, URL | ONE         | Annotation identifier                                           |

**Recommended Properties:**

| Property         | Expected Type | Cardinality | Description                                                       |
| ---------------- | ------------- | ----------- | ----------------------------------------------------------------- |
| `name`           | Text          | ONE         | Name of the annotation (e.g., "Active site", "FMN binding")       |
| `additionalType` | URL           | MANY        | Nature of annotation (e.g., domain, active site, variant)         |
| `description`    | Text          | ONE         | Description of the annotated feature                              |
| `creationMethod` | PropertyValue | ONE         | Method used to generate the annotation (experimental / predicted) |
| `url`            | URL           | ONE         | Link to annotation in database                                    |

**Optional Properties (MIX-MB(M) relevant):**

| Property                | Expected Type            | Cardinality | Description                                  |
| ----------------------- | ------------------------ | ----------- | -------------------------------------------- |
| `hasCategoryCode`       | CategoryCode             | MANY        | Controlled vocabulary term (e.g., EC number) |
| `location`              | PropertyValue, Text, URL | MANY        | Position in sequence (residue number, range) |
| `isPartOfBioChemEntity` | BioChemEntity, URL       | MANY        | Protein this annotation belongs to           |
| `subcellularLocation`   | URL                      | MANY        | Subcellular compartment                      |
| `additionalProperty`    | PropertyValue            | MANY        | Extra annotation details                     |

***

## 3. Ontologies and Standards

### 3.1 NCBI Taxonomy

**Mandatory Requirements:**

* **NCBI TaxID:** Unique numerical identifier for each organism
* **Scientific Name:** Full binomial name (Genus species)
* **Taxonomic Lineage:** Complete classification path
  * Domain (Bacteria, Archaea, Eukarya)
  * Phylum
  * Class
  * Order
  * Family
  * Genus
  * Species

**Example:**

```yaml
organism:
  ncbi_taxid: 1351
  scientific_name: "Enterococcus faecalis"
  lineage:
    domain: "Bacteria"
    phylum: "Firmicutes"
    class: "Bacilli"
    order: "Lactobacillales"
    family: "Enterococcaceae"
    genus: "Enterococcus"
    species: "Enterococcus faecalis"
```

**Strain Level Information:**

```yaml
strain:
  strain_id: "ATCC 19433"
  strain_designation: "V583"
  type_strain: false
  genome_sequenced: true
  genome_assembly: "GCA_000007785.1"

  
```

### 3.2 Additional Ontology Requirements

* **Assay Ontology (BAO):** Biotransformation assay types
  * BAO:0000697 (metabolism assay)
  * BAO:0002989 (biotransformation assay)
* **Unit Ontology (UO):** Standardized units
  * UO:0000062 (molar concentration unit)
  * UO:0000031 (minute)
  * UO:0000027 (degree Celsius)
* **NCBI Taxonomy:** Microbial strain identification
  * Use NCBI TaxID for all organisms
  * Include strain information where applicable

### 3.3 MIxS (Minimum Information about any Sequence)

Compliance with [Genomic Standards Consortium (GSC)](https://www.gensc.org/pages/standards-intro.html) MIxS standards:

**Core MIxS Fields:**

* `source_mat_id`: Unique sample identifier
* `sample_collect_device`: Collection method
* `isol_growth_condt`: Isolation and growth conditions
* `cult_root_med`: Culture rooting medium
* `host_subject_id`: Host organism (if applicable)
* `isolation_source`: Source of isolation (e.g., human gut, soil)

**Environmental Context (MIxS-Env):**

* `env_broad_scale`: Biome (using ENVO terms)
* `env_local_scale`: Local environmental features
* `env_medium`: Material in which organism lives

**Example:**

```yaml
mixs_compliance:
  source_mat_id: "Sample_001_GutMicrobiome"
  isolation_source: "human fecal sample"
  env_broad_scale: "ENVO:00002018 (human-associated habitat)"
  env_local_scale: "ENVO:00002043 (gastrointestinal tract)"
  env_medium: "ENVO:00002003 (fecal material)"
  geo_loc_name: "Germany: Leipzig"
  collection_date: "2023-06-15"
  lat_lon: "51.3397° N, 12.3731° E"

  
```

### 3.4 Environment Ontology (ENVO)

**Required for Habitat Description:**

* **Biome:** ENVO biome classification
* **Environmental Feature:** Specific habitat
* **Environmental Material:** Substrate material

**Common ENVO Terms:**

```
Human-associated environments:
- ENVO:00002043 - gastrointestinal tract
- ENVO:00002042 - oral cavity
- ENVO:00002045 - skin

Environmental sources:
- ENVO:00002007 - sediment
- ENVO:00001998 - soil
- ENVO:00002019 - freshwater environment
- ENVO:00002030 - aquatic environment
```

### 3.5 Phenotype Ontology (PATO/OMP)

**Microbial Phenotype Characteristics:**

* **Gram stain:** PATO:0001654 (gram-positive) or PATO:0001655 (gram-negative)
* **Cell shape:** PATO terms for morphology
* **Motility:** OMP:0000001 (motile) vs non-motile
* **Spore formation:** Presence/absence
* **Oxygen requirement:**
  * Aerobic (PATO:0001903)
  * Anaerobic (PATO:0001904)
  * Facultative (PATO:0002224)

**Example:**

```yaml
phenotype:
  gram_stain: "PATO:0001655 (gram-negative)"
  cell_shape: "rod-shaped"
  motility: "motile"
  oxygen_requirement: "PATO:0002224 (facultative anaerobe)"
  spore_formation: false
  flagella: true
```

### 3.6 Gene Ontology (GO) & Enzyme Commission (EC)

**For Biotransformation Genes/Enzymes:**

* **GO Terms:** Molecular function, biological process
* **EC Numbers:** Enzyme classification
* **UniProt ID:** Protein identification

**Example:**

```yaml
enzyme_information:
  gene_name: "porA"
  locus_tag: "EF_1234"
  go_terms:
    - "GO:0016491 (oxidoreductase activity)"
    - "GO:0009058 (biosynthetic process)"
  ec_number: "EC 1.1.1.1"
  uniprot_id: "P12345"
  function: "Nitroreductase involved in drug metabolism"
```

### 3.7 Antibiotic Resistance Ontology (ARO)

**For Clinical/Environmental Strains:**

```yaml
antibiotic_resistance:
  profile: "ampicillin resistant, chloramphenicol sensitive"
  aro_terms:
    - "ARO:3000007 (beta-lactamase resistance)"
  resistance_genes:
    - gene: "blaZ"
      mechanism: "antibiotic inactivation"
```

***

## 7. Controlled Vocabularies

### 7.1 Oxygen Requirements

Standard terms:

* `obligate aerobe` - Requires oxygen
* `facultative anaerobe` - Grows with or without oxygen
* `obligate anaerobe` - Cannot tolerate oxygen
* `microaerophile` - Requires low oxygen (2-10%)
* `aerotolerant anaerobe` - Tolerates oxygen but doesn't use it

### 7.2 Cell Morphology

* `rod` / `bacillus` - Rod-shaped
* `coccus` - Spherical
* `spirillum` - Spiral
* `vibrio` - Comma-shaped
* `filamentous` - Long filaments
* `pleomorphic` - Variable shapes

### 7.3 Sample Types

* `pure culture` - Single strain isolation
* `enrichment culture` - Enriched for specific function
* `mixed culture` - Defined co-culture
* `community` - Complex microbial community
* `cell-free lysate` - Disrupted cells
* `membrane fraction` - Isolated membranes
* `cytoplasmic fraction` - Soluble proteins

### 7.4 Growth Phases

* `lag phase` - Adaptation period
* `exponential phase` / `log phase` - Rapid growth
* `stationary phase` - Growth plateau
* `death phase` - Cell death exceeds growth
* `early exponential` - Beginning of log phase (OD₆₀₀ 0.2-0.4)
* `mid exponential` - Middle log phase (OD₆₀₀ 0.4-0.8)
* `late stationary` - Extended stationary (>24h)

***

## 4. Data Validation Rules

Before submitting data, verify that all assay records pass the following checks. The pipeline (`generate_assay.R`) performs these checks automatically; they also apply to manually prepared submissions.

### 4.1 Organism Identifiers

| Rule                       | Check                                                                                             |
| -------------------------- | ------------------------------------------------------------------------------------------------- |
| NCBI TaxID is present      | Every assay row must have a non-empty `ASSAY_TAX_ID`                                              |
| NCBI TaxID is numeric      | Must be a positive integer (e.g. `1351`, not `"Enterococcus"`)                                    |
| Scientific name is present | `ASSAY_ORGANISM` must contain the full binomial name (Genus species)                              |
| Name–TaxID consistency     | The scientific name must match the registered name for that TaxID in NCBI Taxonomy                |
| Novel isolates             | If no strain-level TaxID exists, use the species-level TaxID and add a note in `ACTIVITY_COMMENT` |

### 4.2 Assay Index (AIDX)

| Rule            | Check                                                                                                            |
| --------------- | ---------------------------------------------------------------------------------------------------------------- |
| AIDX is present | Every row in `ASSAY.tsv` must have an AIDX                                                                       |
| AIDX format     | Must follow `[Author]_[Genus]_[species]_[Condition]`, using only ASCII letters, digits, underscores, and hyphens |
| AIDX uniqueness | Each AIDX must be unique within the study                                                                        |
| No spaces       | Spaces are not permitted in AIDX values                                                                          |

### 4.3 Required Fields

The following fields must be non-empty in `ASSAY.tsv` for every assay record:

* `AIDX`, `RIDX`, `ASSAY_DESCRIPTION`, `ASSAY_TYPE`, `ASSAY_ORGANISM`, `ASSAY_TAX_ID`

### 4.4 Controlled Vocabulary Terms

| Field                | Allowed values                                                                                                                                    |
| -------------------- | ------------------------------------------------------------------------------------------------------------------------------------------------- |
| `ASSAY_TYPE`         | `bacteria`, `enzyme`, `community`                                                                                                                 |
| `oxygen_requirement` | `obligate aerobe`, `facultative anaerobe`, `obligate anaerobe`, `microaerophile`, `aerotolerant anaerobe` (Section 7.1)                           |
| `sample_type`        | `pure culture`, `enrichment culture`, `mixed culture`, `community`, `cell-free lysate`, `membrane fraction`, `cytoplasmic fraction` (Section 7.3) |
| `harvest_phase`      | `lag phase`, `exponential phase`, `stationary phase`, `death phase`, `early exponential`, `mid exponential`, `late stationary` (Section 7.4)      |

### 4.5 `sameAs` Linking

| Rule                 | Check                                                                       |
| -------------------- | --------------------------------------------------------------------------- |
| NCBI Taxonomy URL    | Required for all organisms with a registered TaxID (Gold tier)              |
| No speculative links | Do not add `sameAs` for novel isolates without a registered external record |

***

## 5. Data Quality Tiers

MIX-MB(M) defines three compliance tiers for microbial organism data. Higher tiers enable broader reuse and interoperability with genomic and phenotypic databases.

### Tier 1 — Gold (Publication-Ready)

Meets all mandatory and recommended fields. Suitable for ChEMBL submission and FAIR data publications.

* NCBI TaxID present and verified
* Full binomial scientific name matching the TaxID
* Strain designation provided (e.g. ATCC, DSMZ, or culture collection number)
* Genome assembly accession available (`GCA_` or `GCF_` accession)
* `sameAs` link to NCBI Taxonomy URL provided; LPSN link included for prokaryotes
* All growth conditions documented: medium, temperature, oxygen requirement, harvest phase
* Full Bioschemas Taxon + Sample JSON-LD records (Sections 2.1 and 2.3)

### Tier 2 — Silver (Research-Grade)

Meets all mandatory fields with partial recommended fields. Suitable for internal data sharing and preprint deposition.

* NCBI TaxID present and verified
* Full binomial scientific name
* Strain designation provided
* `sameAs` link to NCBI Taxonomy URL
* Basic growth conditions: at minimum, medium and temperature

### Tier 3 — Bronze (Preliminary / Screening)

Meets only mandatory fields. Suitable for large-scale screening data where full strain characterisation is not yet available.

* NCBI TaxID present
* Scientific name present
* `AIDX` and `RIDX` assigned
* `ASSAY_TYPE` specified
* Minimal cultivation information; quantitative growth conditions not required

***

## 6. How to use the Template

The MIX-MB submission template is provided as [Templates/Template.xlsx](https://github.com/zmahnoor14/BioXend/blob/devel/Standards/Templates/Template.xlsx). The **Assay** sheet covers the MIX-MB(M) standard.

### Colour coding

| Colour | Meaning                                                       |
| ------ | ------------------------------------------------------------- |
| Green  | Mandatory — must be filled for a valid submission             |
| Blue   | Recommended — strongly encouraged; required for Tier 1 (Gold) |
| Yellow | Optional — fill if available                                  |

### Step-by-step

1. **Open** `Template.xlsx` and go to the **Assay** sheet.
2. **Add one row per organism × condition combination.** If the same organism is used under two different oxygen conditions or media, it gets two rows with distinct AIDXs.
3. **Look up the NCBI TaxID** for each organism:
   * Go to <https://www.ncbi.nlm.nih.gov/taxonomy>
   * Search by scientific name; copy the TaxID number into `ASSAY_TAX_ID`
4. **Assign the AIDX** using the format `[FirstAuthorLastName]_[Genus]_[species]_[Condition]`, e.g. `Zimmermann_Actinomyces_graevenitzii_biotransformation`.
5. **Fill in cultivation conditions** (medium, temperature, oxygen requirement) using the controlled vocabulary terms from Section 7.
6. **For community assays,** set `ASSAY_ORGANISM` to `mixed microbial community`, `ASSAY_TAX_ID` to `1` (root), and describe the community source in `ASSAY_DESCRIPTION`.
7. **Fill in `ASSAY_PARAM.tsv`** for quantitative experimental parameters (temperature, incubation time, pH, cell density, substrate concentration). Each parameter gets its own row linked by AIDX.
8. **Validate** your completed sheet against the rules in Section 4 before running the pipeline or submitting.

***

## 7. Version History

| Version | Date       | Changes |
| ------- | ---------- | ------- |
| 0.1.0   | 2026-02-05 | Draft   |

***

## 8. Data Access and Repository Guidance

### 8.1 Repository and Standard Documents

The MIX-MB standard documents, submission template, and BioXend pipeline are openly available:

* **Standard documents:** [GitHub — zmahnoor14/BioXend](https://github.com/zmahnoor14/BioXend), under `Standards/`
* **Submission template:** `Standards/Templates/Template.xlsx` (see Section 6)
* **BioXend pipeline:** Nextflow workflow (`main.nf`) in the repository root

### 8.2 Files Generated by This Standard

MIX-MB(M) organism data feed the assay files required for ChEMBL deposition. The BioXend pipeline (`generate_assay.R`) produces:

| Output File       | Content                                                                               |
| ----------------- | ------------------------------------------------------------------------------------- |
| `ASSAY.tsv`       | Assay descriptions per organism × condition, indexed by AIDX                          |
| `ASSAY_PARAM.tsv` | Quantitative experimental parameters (temperature, pH, incubation time, cell density) |

These files are linked to compound and activity records via AIDX (see MIX-MB(B)).

### 8.3 Submitting to ChEMBL

MIX-MB(M) assay files are deposited to [ChEMBL](https://www.ebi.ac.uk/chembl/) as part of a full MIX-MB submission package. For deposition enquiries, contact **<chembl-help@ebi.ac.uk>** or use the ChEMBL deposition portal.

**Pre-submission checklist for organism data:**

* All assay rows pass Section 4 validation rules
* Every `ASSAY_TAX_ID` is a valid, numeric NCBI TaxID
* Scientific name matches the registered NCBI name for that TaxID
* At least one `sameAs` URL to NCBI Taxonomy (Gold tier)

### 8.4 Supplementary Sequence Data Repositories

Sequencing data linked to strains used in a MIX-MB study should be deposited in:

| Repository    | URL                                     | Data type                              |
| ------------- | --------------------------------------- | -------------------------------------- |
| NCBI SRA      | <https://www.ncbi.nlm.nih.gov/sra>      | Short-read and long-read sequencing    |
| ENA           | <https://www.ebi.ac.uk/ena>             | Alternative / European submission      |
| NCBI Assembly | <https://www.ncbi.nlm.nih.gov/assembly> | Genome assemblies (GCA/GCF accessions) |

Record the assembly accession in `additionalProperty` → `genome_assembly` (Section 2.3) and include it in the `sameAs` field of the Taxon record.

***

## 9. Licence and Reuse

### 9.1 Standard Documents

The MIX-MB standard documents are released under the **Creative Commons Attribution 4.0 International (CC BY 4.0)** licence (<https://creativecommons.org/licenses/by/4.0/>).

You are free to share, adapt, and build upon this standard for any purpose, provided you give appropriate credit:

> Zulfiqar M. *et al.* MIX-MB: Minimum Information about Xenobiotics-Microbiome Biotransformation (v0.1.0). GitHub: <https://github.com/zmahnoor14/BioXend>

### 9.2 Pipeline and Code

The BioXend Nextflow pipeline and associated scripts are released under the **MIT Licence**. See `LICENSE` in the repository root for full terms.

### 9.3 Submitted Data

Data deposited using MIX-MB formats in public repositories are subject to that repository's terms:

| Repository          | Licence                                                                      |
| ------------------- | ---------------------------------------------------------------------------- |
| ChEMBL              | CC BY-SA 3.0 (<https://creativecommons.org/licenses/by-sa/3.0/>)             |
| NCBI SRA / Assembly | NCBI data use policies (<https://www.ncbi.nlm.nih.gov/home/about/policies/>) |
| ENA                 | EMBL-EBI data access policies                                                |

When reusing MIX-MB-formatted datasets, cite both the original study (via its RIDX/DOI) and the MIX-MB standard.

***

## 10. Provenance

Provenance records the origin, history, and chain of custody of microbial organism data reported under MIX-MB(M). Complete provenance supports the **Reusable** principle of FAIR and enables reproducibility of biotransformation experiments.

### 10.1 Study-Level Provenance

Every assay record is anchored to a **Reference record** (RIDX) in `REFERENCE.tsv`:

| Field                      | Description                                         |
| -------------------------- | --------------------------------------------------- |
| `RIDX`                     | Study-local identifier linking all submission files |
| `DOI`                      | Digital Object Identifier of the source publication |
| `TITLE`, `AUTHORS`, `YEAR` | Full bibliographic metadata                         |

The RIDX appears in every row of `ASSAY.tsv`, making the publication the root provenance node for all organism data.

### 10.2 Strain Identity Provenance

The organism identifiers documented in Section 1.4 serve as provenance anchors for strain identity:

| Identifier                    | Provenance Source                                                                  |
| ----------------------------- | ---------------------------------------------------------------------------------- |
| **NCBI TaxID**                | Verified against NCBI Taxonomy; links to authoritative taxonomy record             |
| **LPSN name**                 | Valid scientific name from the List of Prokaryotic Names (<https://lpsn.dsmz.de/>) |
| **Culture collection ID**     | Physical strain held at a registered biobank (ATCC, DSMZ, CGSC, etc.)              |
| **Genome assembly accession** | Sequenced genome lodged in NCBI Assembly or ENA                                    |
| `sameAs` URLs                 | Direct links to NCBI Taxonomy, LPSN, BacDive, or other authoritative records       |

For novel isolates without a registered TaxID, the closest assigned TaxID plus a note in `ACTIVITY_COMMENT` constitutes the strain's provenance record until formal registration.

### 10.3 Environmental and Isolation Provenance

For strains isolated from environmental or clinical samples, document the following MIxS-compliant fields (Section 3.3) as part of strain provenance:

| Field                                 | Provenance Aspect                                                     |
| ------------------------------------- | --------------------------------------------------------------------- |
| `isolation_source`                    | Biological or environmental matrix from which the strain was obtained |
| `collection_date`                     | ISO 8601 date of sample collection                                    |
| `geo_loc_name`                        | Geographic origin of the sample                                       |
| `env_broad_scale` / `env_local_scale` | ENVO-annotated habitat classification                                 |

### 10.4 Pipeline Provenance

When generating submission files with BioXend, record in `REFERENCE.tsv` or `ASSAY_PARAM.tsv`:

* **BioXend pipeline version** — from `versions/pipeline.txt`
* **`generate_assay.R` version** — from the script header
* **Date of generation** — ISO 8601 format (YYYY-MM-DD)

***

## 11. References

1. NCBI Taxonomy Database: <https://www.ncbi.nlm.nih.gov/taxonomy>
2. Genomic Standards Consortium (GSC): <https://www.gensc.org/>
3. MIxS Standards: <https://press3.mcs.anl.gov/gensc/mixs/>
4. Bioschemas Specifications: <https://bioschemas.org/>
5. Environment Ontology (ENVO): <http://www.environmentontology.org/>
6. LPSN - List of Prokaryotic names: <https://lpsn.dsmz.de/>
7. BacDive - Bacterial Diversity Database: <https://bacdive.dsmz.de/>
8. FAIR Principles: <https://www.go-fair.org/fair-principles/>
9. ChEMBL Database: <https://www.ebi.ac.uk/chembl/>

***

## 12. Contact and Contributions

For questions, suggestions, or contributions to this standard, please contact:

* **Maintainer:** Mahnoor Zulfiqar
* **Institution:** NFDI4Microbiota
* **Email:** [Contact information](mailto:zmahnoor14@gmail.com)
* **Repository:** \[[GitHub repository URL](https://github.com/zmahnoor14/BioXend)]

***


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