🌱 Environmental Biotechnology — Micro-pollutant Degradation (Theory Summary)

(Day 11 Part 2 — Complete Theoretical Summary)

⭐ 1. Why studying micro-pollutant degradation is difficult

Micro-pollutants (pharmaceuticals, hormones, pesticides, etc.) occur at extremely low concentrations and often in many different chemical forms.

Why gene-based detection fails for micro-pollutants

For nutrients like nitrogen or phosphorus, we know:

• the enzymes required to degrade them
• the genes encoding those enzymes

So we can simply search for the right genes.

But for micro-pollutants:

• pathways are unknown
• enzymes and genes are not documented
• databases do not contain the relevant sequences

👉 We cannot search for genes when we don’t know what to look for. So we need alternative, activity-based methods.

⭐ 2. Community analysis: what we can detect vs what we can’t

2.1 16S rRNA (amplicon) sequencing 🧬

This gives:

• taxonomic composition (relative abundance)
• high-resolution identification But it does not show activity, only presence.

2.2 Imaging approaches (FISH) 🔬

Fluorescent probes bind to specific rRNA sequences.

Advantages:

• single-cell quantification
• can assign cells to functional groups (e.g., nitrifiers, denitrifiers) Drawbacks:
• limited by available probes
• slower than sequencing
• lower taxonomic resolution

“Others” group and “tourists” 👤🧳

15–20% of cells in wastewater cannot be matched with any known probe. They are called tourists because:

• they appear only temporarily
• their function is unknown
• they cannot be assigned to metabolic roles

Understanding this group is important because micro-pollutant degradation often occurs in rare, unknown bacteria.

⭐ 3. MAR-FISH: Microautoradiography + FISH

A method invented to directly link identity + activity.

How it works ⚛️

1. Incubate sample with a radioactively labelled substrate (e.g., ¹⁴C-EE2).
2. Cells that metabolize the substrate incorporate radioactive carbon.
3. Cells are placed on a slide and covered with silver-bromide photographic emulsion.
4. Radioactivity reduces silver → black silver grains appear above active cells.
5. Combine with FISH probes → identity + function overlay.

What this reveals

• Only a microscopic fraction of cells (e.g., 0.1‰) degrade a specific micro-pollutant.
• These rare organisms are extremely efficient despite tiny population sizes.
• They often come from the “others”/tourist fraction.

This is one of the first technologies allowing: “This specific bacterium degraded this specific compound.”

⭐ 4. Stable Isotope Probing (SIP) 🌀

A faster, high-throughput alternative to MAR-FISH.

Principle

Use substrates labelled with 13C (heavy carbon) instead of 12C.

Process:

1. Incubate community with a mixture of ¹²C and ¹³C substrate.
2. If organisms degrade the compound, they incorporate ¹³C into their biomass → ¹³C-DNA becomes heavier.
3. Ultracentrifuge in cesium chloride gradient (100,000 g, ~3 days).
4. Heavy DNA forms a separate band.
5. Sequence the heavy fraction → reveals which organisms used the compound.

Strengths

• detects hundreds of active degraders simultaneously
• identifies functional players without prior gene knowledge

Limitations

• does not show micro-scale localization
• requires costly ultracentrifugation

⭐ 5. Biometer flasks & radiolabelled mineralization assays 🧪

An old but powerful approach to measure mineralization of compounds.

How it works

• Provide substrate labelled with ¹⁴C.
• If bacteria fully degrade the compound → CO₂ is produced.
• A CO₂ trap (alkaline NaOH solution) captures the CO₂.
• Measure radioactive CO₂ in a scintillation counter.

What this tells you

• How much of the compound is mineralized.
• Detection is extremely sensitive.

What it cannot tell you

• Which organisms performed the degradation.

It is purely quantitative, not taxonomic.

⭐ 6. Toxicological effect-based detection 🐟

Chemical detection (e.g., GC-MS) only finds known parent molecules. But humans often modify pharmaceuticals before excretion (metabolites). So the chemical signature may not match the database → compound is missed.

Effect-based bioassays

Use living organisms that respond to pollutants:

• fish swimming behavior
• Daphnia mobility
• avoidance/attraction responses

Example: Fish exposed to Prozac swim hyperactively → you detect effect, not chemical structure.

Advantages:

• detects transformation products
• reveals ecological impact

Limitations:

• qualitative or semi-quantitative
• no molecular identification

⭐ 7. Membrane bioreactors (MBR) & carrier systems (“MBBR”) 🧱🦠

Why normal wastewater systems fail

Micro-pollutant degraders are:

• rare
• slow-growing
• easily washed out

Adding carriers (biofilm supports)

Plastic carriers provide surfaces for bacteria to attach and stay. This retains rare degraders and prevents washout.

Membrane bioreactors

Membranes act as:

• physical retention barriers
• high-surface-area habitats

Effect

Removal efficiency increases 8–10× for many pollutants. Example: Diclofenac

• almost no removal in conventional activated sludge
• strong removal when biofilms or membranes are used

Reason: Biofilms retain specialist degraders long enough to perform slow transformations.

⭐ 8. Bioaugmentation 🧫➕🦠

Adding specialized degraders into a system.

Why it sounds good

Companies sell bacteria advertised to degrade:

• oils
• pesticides
• pharmaceuticals

Why it often fails (theory)

1. Enrichment strain weakness Lab-enriched strains become:
   • fat
   • slow
   • uncompetitive compared to natural wastewater microbes.
2. Indigenous community resistance Native bacteria produce antibiotics to defend their niche. Enriched strains lack resistance and lose the competition.
3. Grazing by protists Non-biofilm bacteria are easily eaten by protozoa.
4. Phage infections High density of identical added bacteria → phages spread easily.

Outcome

Bioaugmented strains usually survive only hours to days. Thus the effect is:

• short-lived
• rarely stable
• often not worth the cost

Bioaugmentation rarely succeeds in open, competitive systems.