Environmental microbiology

Microbiology (B.Sc) · Applied microbiology · free study notes for exams, placements and interviews

In short

Environmental microbiology studies microorganisms in soil, water and air — how they cycle elements, degrade pollutants, indicate contamination and are harnessed to clean waste. It underpins sewage and effluent treatment, drinking-water safety testing, bioremediation of oil and pesticide spills, and the biogeochemical cycles that keep soils fertile. In Indian B.Sc and M.Sc syllabi (UGC CBCS) it carries large marks on biogeochemical cycles, water quality indicators and wastewater treatment; it also appears in CSIR-UGC NET Life Sciences, GATE Biotechnology (XL/BT) and ICAR exams. Employers who test it include CPCB and State Pollution Control Boards, municipal and water-board labs, NABL-accredited testing houses (SGS, TÜV, Eurofins), ETP/STP operators and EIA consultancies.

Explained

Microbial habitats and ecology

Environment compartments: soil (highest diversity, ~10⁸–10⁹ bacteria/g), freshwater, marine, air (aerosols, no permanent flora), and extreme habitats.
Rhizosphere: soil zone influenced by root exudates; microbial counts 10–100× bulk soil (R:S ratio). Hosts *Rhizobium*, *Azotobacter*, *Pseudomonas*, mycorrhizae.
Terms: *habitat* (physical place), *niche* (functional role), *microbial consortium* (co-operating mixed species), *syntrophy* (one organism's product is another's substrate — key in anaerobic digestion).
Interactions: mutualism (lichen = alga/cyanobacterium + fungus; *Rhizobium*–legume), commensalism, synergism, competition, amensalism (antibiosis), parasitism, predation (protozoa grazing bacteria in activated sludge).
Biofilm: surface-attached community in an EPS (exopolysaccharide) matrix; formed by attachment → EPS production → maturation → dispersal. Gives 10–1000× antibiotic/biocide tolerance; basis of trickling filters and MBBR, but also pipe biofouling and biocorrosion.
Extremophiles: thermophiles (*Thermus*), hyperthermophiles (*Pyrococcus*), acidophiles (*Acidithiobacillus*, pH <2), alkaliphiles, halophiles (*Halobacterium*), psychrophiles, barophiles. Sources of industrial enzymes (Taq polymerase, thermostable lipases).
VBNC and the "great plate count anomaly": only ~0.1–1% of environmental bacteria grow on plates, so culture-independent methods (16S rRNA amplicon sequencing, metagenomics, FISH, DGGE, qPCR) are used for community profiling.

Biogeochemical cycles

Carbon cycle: CO₂ fixed by photoautotrophs (Calvin cycle) and chemoautotrophs; returned by respiration, fermentation and methanogenesis. Methanogens (Archaea) produce CH₄ in paddy fields, rumen and landfills; methanotrophs (*Methylococcus*) oxidise it back.
Nitrogen cycle steps:
Nitrogen fixation — N₂ → NH₃ by nitrogenase; symbiotic (*Rhizobium*, *Bradyrhizobium*, *Frankia*), free-living aerobic (*Azotobacter*), anaerobic (*Clostridium*), cyanobacterial (*Anabaena* in *Azolla*, paddy fields), associative (*Azospirillum*).
Ammonification — organic N → NH₄⁺ by proteolytic/deaminating microbes.
Nitrification — NH₄⁺ → NO₂⁻ (*Nitrosomonas*) → NO₃⁻ (*Nitrobacter*, *Nitrospira*); strictly aerobic chemolithotrophs, slow growers, inhibited below pH 6 and by heavy metals; comammox (*Nitrospira inopinata*) does both steps.
Denitrification — NO₃⁻ → NO₂⁻ → NO → N₂O → N₂ by facultative anaerobes (*Pseudomonas*, *Paracoccus*); needs anoxic conditions and an organic carbon source — the basis of biological nitrogen removal.
Anammox — NH₄⁺ + NO₂⁻ → N₂ by *Brocadia*/*Kuenenia*; no organic carbon and ~60% less aeration needed.
Nitrogenase is O₂-sensitive; legume nodules protect it with leghaemoglobin, heterocysts protect it in cyanobacteria.
Sulphur cycle: H₂S → S⁰ → SO₄²⁻ by *Beggiatoa*, *Thiobacillus*/*Acidithiobacillus*, and photosynthetic *Chlorobium*/*Chromatium*; sulphate reduction to H₂S by *Desulfovibrio* (causes black sludge, concrete corrosion in sewers, souring of oil wells).
Phosphorus cycle: no gaseous phase; phosphate-solubilising bacteria (*Bacillus megaterium*, *Pseudomonas striata*) and fungi (*Aspergillus*, *Penicillium*) release organic acids and phytase to free bound P.
Iron and manganese: *Gallionella*, *Leptothrix* oxidise Fe²⁺ → Fe³⁺ causing red water and clogged bore wells; *Acidithiobacillus ferrooxidans* drives acid mine drainage and bioleaching.

Water quality, indicators and testing

Indicator organism criteria: present whenever the pathogen is, non-pathogenic, more numerous and more persistent than pathogens, easy to detect, absent from unpolluted water.
Indicators used: total coliforms (screening), thermotolerant/faecal coliforms, *E. coli* (definitive faecal marker), faecal streptococci/enterococci (persist longer), *Clostridium perfringens* spores (old pollution), coliphages (viral surrogate).
Methods: MPN / multiple-tube fermentation (presumptive in lactose/MacConkey broth with Durham tube → confirmed in BGLB → completed on EMB/endo agar; Eijkman test at 44.5 °C for *E. coli*); membrane filtration (0.45 µm, count colonies on m-Endo/m-FC — results in cfu/100 mL); enzyme substrate tests (ONPG for β-galactosidase, MUG for β-glucuronidase); H₂S strip test as a field screen.
IS 10500:2012 (Drinking Water Specification, with amendments): coliform bacteria shall be absent in any 100 mL sample; *E. coli* absent; no relaxation permitted for microbiological requirements. Also turbidity ≤1 NTU (acceptable), residual free chlorine ≥0.2 mg/L at the consumer end (0.5 mg/L recommended in epidemics).
Potability determinants: Eh, DO, BOD, COD, TOC, turbidity, nitrate, coliform count. Sanitary survey of the source accompanies lab testing.
BOD = O₂ consumed by microbes degrading organic matter, measured as BOD₅ at 20 °C (Indian practice often reports BOD₃ at 27 °C ≈ 0.8–0.9 × BOD₅). COD = chemical oxidation by acidified K₂Cr₂O₇ with Ag₂SO₄ catalyst and HgSO₄ to mask chlorides. COD > BOD always; BOD/COD >0.5 means readily biodegradable, <0.3 means largely refractory.
Eutrophication: nutrient (N, P) enrichment → algal bloom → death and decomposition → O₂ depletion → fish kill; cyanobacterial toxins (microcystin). P is usually the limiting nutrient in freshwater.
Waterborne diseases: cholera (*Vibrio cholerae*), typhoid (*Salmonella* Typhi), shigellosis, hepatitis A and E, polio, rotavirus, giardiasis, amoebiasis, cryptosporidiosis (chlorine-resistant oocysts — needs filtration/UV).

Wastewater treatment

Stages: preliminary (screening, grit removal) → primary (sedimentation, removes ~30% BOD, 60% SS) → secondary/biological (removes 80–95% BOD) → tertiary (nutrient removal, filtration, disinfection) → sludge handling.
Aerobic suspended growth — activated sludge process (ASP): aeration tank + secondary clarifier with return activated sludge (RAS); biomass measured as MLSS/MLVSS; controlled by F/M ratio, SRT (sludge age), HRT and DO (≥2 mg/L). Floc = bacteria (*Zoogloea*), protozoa (ciliates indicate healthy sludge), rotifers.
Sludge problems: bulking by filamentous *Microthrix*/*Nocardia*/*Sphaerotilus* (low F/M, low DO, nutrient deficiency) — diagnosed by SVI (>150 mL/g = bulking); foaming by *Nocardia*; rising sludge from denitrification in the clarifier; pin floc from over-oxidation.
Variants: extended aeration, oxidation ditch, SBR (sequencing batch reactor — fill, react, settle, decant, idle; widely used in Indian STPs), MBR (membrane bioreactor — high quality permeate for reuse), MBBR.
Aerobic attached growth: trickling filter (rock/plastic media, zoogleal film, recirculation), rotating biological contactor (RBC).
Anaerobic treatment: four phases — hydrolysis → acidogenesis → acetogenesis → methanogenesis; biogas ~60–70% CH₄, 30–40% CO₂. UASB reactors (granular sludge blanket) are the standard for Indian sewage and distillery spent wash; also anaerobic digesters for sludge stabilisation, fixed-film and anaerobic filters. Low sludge yield, energy positive, but slow start-up, poor pathogen removal and needs post-treatment polishing ponds.
Natural systems: waste stabilisation ponds (anaerobic → facultative → maturation; algal–bacterial symbiosis supplies O₂), aerated lagoons, constructed wetlands (*Typha*, *Phragmites*, Canna), root-zone treatment, duckweed/fish ponds.
Nutrient removal: nitrification–denitrification (MLE, A2/O, Bardenpho), EBPR using polyphosphate-accumulating organisms (*Accumulibacter*) cycled through anaerobic/aerobic zones.
Disinfection: chlorination (contact time; forms THMs), UV (no residual, used in most new Indian STPs for reuse), ozone, chlorine dioxide.
Regulation: Water (Prevention and Control of Pollution) Act, 1974; Air Act, 1981; Environment (Protection) Act, 1986 and its Rules; Hazardous and Other Wastes Rules, 2016; Solid Waste Management Rules, 2026 (replaced the 2016 Rules from 1 April 2026); Bio-Medical Waste Management Rules, 2016. Consent to Establish/Operate from the State Pollution Control Board; CPCB sets effluent standards (general inland-surface-water discharge limits BOD 30 mg/L, COD 250 mg/L, SS 100 mg/L — sector-specific and STP norms differ and have been revised by notification, so check the current schedule). NGT hears environmental disputes.

Bioremediation and solid waste

Definition: use of microbes, plants or their enzymes to detoxify or remove pollutants.
Strategies: in situ — natural attenuation, biostimulation (add N, P, O₂, electron acceptors), bioventing, biosparging, bioaugmentation (add specialist strains); ex situ — landfarming, biopiles, composting, slurry bioreactors.
Xenobiotics: recalcitrant compounds — DDT, PCBs, dioxins, PAHs, pesticides, dyes, plastics. Degraders: *Pseudomonas putida* (toluene, naphthalene; TOL plasmid), *Alcaligenes*, *Mycobacterium*, *Phanerochaete chrysosporium* (lignin peroxidase, degrades dyes/PAHs). Co-metabolism degrades a compound that is not a growth substrate.
Oil spills: hydrocarbon degraders (*Alcanivorax*, *Acinetobacter*) plus oleophilic fertiliser and dispersants. Oilzapper (TERI) — an Indian bacterial consortium used by ONGC/IOC for oily sludge.
Heavy metals: cannot be degraded — only transformed or immobilised: biosorption (dead biomass, chitin, algae), bioaccumulation, biomethylation (Hg), bioprecipitation as sulphides by *Desulfovibrio*, Cr(VI)→Cr(III) reduction, mer operon mercury resistance.
Phytoremediation: phytoextraction, rhizofiltration, phytostabilisation, phytodegradation, phytovolatilisation; *Brassica juncea*, *Helianthus*, water hyacinth, vetiver. Cheap but slow and depth-limited.
Bioleaching: *Acidithiobacillus ferrooxidans*/*thiooxidans* and *Leptospirillum* recover Cu, U, Au via direct and indirect (Fe³⁺ oxidation) mechanisms — dump, heap and in-situ leaching.
Solid waste: aerobic composting (mesophilic → thermophilic 55–65 °C → cooling → maturation; C:N ~25–30:1), vermicomposting (*Eisenia foetida*), windrow/in-vessel systems, biomethanation of wet waste, landfill gas recovery; leachate is high-COD and needs treatment. Sanitation: twin-pit latrines, septic tank + soak pit, faecal sludge management under Swachh Bharat Mission.
Air microbiology: bioaerosols sampled by settle plates, slit-to-agar, Andersen cascade impactor, liquid impingers; HEPA filtration and laminar flow in clean rooms; cleanroom particle classification under ISO 14644, biocontamination control under ISO 14698, and microbial air limits per cleanroom grade under Schedule M pharmaceutical practice.
GEMs: genetically engineered microbes (e.g. superbug for oil degradation, *Pseudomonas* with engineered plasmids) face containment and regulatory limits under the Rules for Manufacture, Use, Import, Export and Storage of Hazardous Microorganisms/GMOs, 1989 (GEAC approval).

Key points

Environmental microbiology — study of microbial roles in soil, water and air, including pollution control and element cycling.
Indicator organism — a harmless organism (coliform, *E. coli*) whose presence signals faecal contamination; IS 10500:2012 requires coliforms absent in 100 mL.
MPN — statistical estimate of coliform density per 100 mL from multiple-tube fermentation; membrane filtration gives a direct cfu count.
BOD₅ — oxygen demand of biodegradable organics at 20 °C over 5 days; a biological measure.
COD — oxygen equivalent of total chemically oxidisable matter; always greater than BOD; BOD/COD indicates biodegradability.
Nitrification — aerobic, two-step oxidation of ammonia by *Nitrosomonas* and *Nitrobacter*; slow, pH and metal sensitive.
Denitrification — anoxic reduction of nitrate to N₂; requires carbon source; removes nitrogen from wastewater.
Nitrogen fixation — nitrogenase reduces N₂ to NH₃; enzyme is oxygen-labile; symbiotic and free-living forms.
Anammox — anaerobic ammonium oxidation with nitrite to N₂; saves aeration and carbon.
Activated sludge — aerated suspended-growth floc with sludge recycle; controlled by F/M, MLSS, SRT and DO.
SVI — settled sludge volume per gram MLSS; above ~150 mL/g indicates filamentous bulking.
UASB — upflow anaerobic sludge blanket; granular biomass, biogas recovery, standard for Indian sewage and distillery effluent.
Anaerobic digestion — hydrolysis, acidogenesis, acetogenesis, methanogenesis yielding 60–70% methane biogas.
Eutrophication — nutrient enrichment causing blooms, oxygen depletion and fish kill; P usually limiting.
Bioremediation — microbial detoxification of pollutants; biostimulation adds nutrients, bioaugmentation adds organisms.
Biosorption — passive binding of heavy metals to biomass; metals are immobilised, never degraded.
Bioleaching — acidophilic Fe/S oxidisers solubilise metals from low-grade ore.
Great plate count anomaly — <1% of environmental microbes are culturable, hence metagenomics and 16S rRNA profiling.

Formulas and examples

BOD (mg/L) = [(DO_i − DO_f) − (B_i − B_f)f] / P — DO_i, DO_f = initial and final dissolved oxygen of the sample bottle (mg/L); B_i, B_f = blank/seed control DO; f = seed volume ratio; P = decimal volume fraction of sample in the bottle. For unseeded samples: BOD = (DO_i − DO_f)/P.

First-order BOD exertion: BOD_t = L₀(1 − e^{−k t}) = L₀(1 − 10^{−K t}) — L₀ = ultimate BOD (mg/L), k = rate constant (per day, base e), K = k/2.303, t = days.

Temperature correction: k_T = k_20 × 1.047^T−20 — T in °C.

F/M ratio (per day) = Q × S₀ / (V × X) — Q = flow (m³/d), S₀ = influent BOD (mg/L), V = aeration tank volume (m³), X = MLVSS (mg/L).

SRT (sludge age, days) = V·X / (Q_w·X_r + Q_e·X_e) — Q_w = waste sludge flow, X_r = RAS solids, Q_e, X_e = effluent flow and solids.

HRT (hours) = V / Q ; Organic loading rate = Q·S₀ / V (kg BOD/m³·d)

SVI (mL/g) = (settled sludge volume in 30 min, mL/L × 1000) / MLSS (mg/L)

Removal efficiency (%) = (S₀ − S_e) × 100 / S₀ — S_e = effluent concentration.

Observed sludge yield: P_x (kg/d) = Y·Q(S₀ − S_e) / [(1 + k_d·SRT) × 1000] — Y = kg VSS/kg BOD, k_d = endogenous decay coefficient (per day).

Specific growth rate: µ = µ_max·S/(K_s + S) (Monod) ; generation time t_g = t·log2 / (log N_t − log N₀)

Example 1 — BOD₅ by dilution. 5 mL of sewage diluted to a 300 mL bottle; DO_i = 8.2 mg/L, DO_f after 5 days = 2.4 mg/L; blank unchanged.

P = 5/300 = 0.01667.
BOD₅ = (8.2 − 2.4)/0.01667 = 5.8 × 60 = 348 mg/L.
Dilution check: depletion 5.8 mg/L lies in the valid 2–6 mg/L window, and residual DO 2.4 > 1 mg/L, so the result is acceptable.

Example 2 — Ultimate BOD and BOD₃ at 27 °C. BOD₅ = 348 mg/L, k₂₀ = 0.23 /day.

1 − e^−0.23×5 = 1 − e^−1.15 = 1 − 0.3166 = 0.6834 → L₀ = 348/0.6834 = 509 mg/L.
k₂₇ = 0.23 × 1.047⁷ = 0.23 × 1.382 = 0.318 /day.
BOD₃,₂₇ = 509 × (1 − e^−0.954) = 509 × (1 − 0.3853) = 509 × 0.6147 = 313 mg/L (≈0.90 × BOD₅, matching the usual Indian conversion).

Example 3 — MPN. Multiple-tube test with 5 tubes each of 10 mL, 1 mL and 0.1 mL gives positives 5-3-1 → MPN index from the standard table = 110 coliforms per 100 mL. Since IS 10500:2012 requires coliforms absent in 100 mL, the water is unfit for drinking without treatment; it would also exceed the designated-best-use Class C limit (≤5000 MPN/100 mL) only if far higher, so the sample is usable as a Class C source after conventional treatment and disinfection.

Example 4 — Activated sludge design check. Q = 5 MLD (5000 m³/d), influent BOD 250 mg/L, effluent 20 mg/L, aeration tank 1250 m³, MLVSS 2500 mg/L, waste sludge 50 m³/d at 10 000 mg/L (effluent solids neglected).

HRT = 1250/5000 = 0.25 d = 6 hours.
BOD load = 5000 × 250/1000 = 1250 kg/d; OLR = 1250/1250 = 1.0 kg BOD/m³·d.
F/M = 1250/(1250 m³ × 2.5 kg/m³) = 1250/3125 = 0.40 /day (typical ASP range 0.2–0.5).
SRT = (1250 × 2500)/(50 × 10 000) = 3 125 000/500 000 = 6.25 days.
Removal efficiency = (250 − 20)×100/250 = 92%; effluent BOD 20 mg/L meets the 30 mg/L inland-discharge limit.

Example 5 — SVI and biogas. Settling test: 30-min settled volume 220 mL/L, MLSS 2800 mg/L.

SVI = 220 × 1000/2800 = 78.6 mL/g → good settling, no bulking (bulking above ~150).
Anaerobic digester receiving 1000 kg COD/d removing 80% at 0.35 m³ CH₄ per kg COD removed: CH₄ = 1000 × 0.8 × 0.35 = 280 m³/d; at 65% CH₄ in biogas, biogas = 280/0.65 ≈ 431 m³/d; energy ≈ 280 × 35.8 MJ/m³ ≈ 10 024 MJ/d (≈2784 kWh/d thermal).

Parameter	Activated sludge (ASP)	UASB (anaerobic)	Waste stabilisation pond
Energy	High (aeration)	Low; produces biogas	Nil
Land need	Low	Low–moderate	Very high (1–2 ha per MLD)
BOD removal	85–95%	65–80% (needs polishing)	70–90%
Sludge produced	High	Low	Very low
Pathogen removal	Moderate (needs disinfection)	Poor	Good in maturation ponds
Operator skill	High	Moderate	Low

Feature	BOD	COD
Basis	Microbial oxidation	Chemical oxidation (dichromate)
Time	5 days (or 3 d at 27 °C)	~3 hours
Covers	Biodegradable only	Biodegradable + refractory
Typical sewage value	200–300 mg/L	400–600 mg/L

Where it is used

Municipal STPs and industrial ETPs/CETPs: process microbiologists and operators run MLSS, SVI, BOD/COD and microscopic floc checks — e.g. Delhi Jal Board, MCGM, NMCG Namami Gange STPs, Tirupur dyeing CETPs.
Water quality and NABL testing labs: coliform MPN/membrane filtration, heterotrophic plate counts, residual chlorine against IS 10500:2012; roles in CPCB/SPCB labs, Jal Jeevan Mission district labs, SGS/Eurofins/TÜV.
Bioremediation and oilfield services: TERI's Oilzapper for ONGC/IOCL/BPCL oily sludge; bioremediation of pesticide-contaminated sites and tannery chrome sludge.
Agri-biologicals: biofertiliser production under FCO (Fertiliser Control Order) specifications — *Rhizobium*, *Azotobacter*, *Azospirillum*, PSB, mycorrhiza; biopesticides (*Bacillus thuringiensis*, *Trichoderma*) by IFFCO, NFL, Kribhco and private units.
Waste-to-energy and compost: biomethanation plants (e.g. Indore, Bengaluru), city compost under Swachh Bharat, GOBARdhan biogas, landfill gas recovery.
EIA, consultancy and audit: environmental consultants preparing EIA/EMP reports, SPCB consent applications and compliance monitoring; mining bioleaching trials (uranium at Jaduguda, copper at Malanjkhand studies).

Interview questions

Q: Why is *E. coli* preferred over detecting *Salmonella* directly in water testing? A: *E. coli* is always present in faeces in large numbers, is easy and cheap to detect, and survives as long as most enteric pathogens, so its absence gives practical assurance that pathogens are absent.
Q: Why is COD always higher than BOD? A: Dichromate oxidises both biodegradable and non-biodegradable organics plus some inorganics, whereas BOD measures only what microbes can oxidise in five days.
Q: What does a high SVI tell a plant operator? A: Sludge is settling poorly, usually due to filamentous bulking from low F/M, low DO or nutrient deficiency; solids then escape with the effluent.
Q: Why does denitrification need an anoxic zone and a carbon source? A: Nitrate is used as a terminal electron acceptor only when O₂ is absent, and the bacteria need organic carbon as electron donor — hence raw sewage or methanol is fed to the anoxic tank.
Q: What is the advantage of UASB over activated sludge for Indian sewage? A: No aeration energy, far less sludge and recoverable biogas; the trade-off is lower BOD removal and poor pathogen removal, so polishing ponds or post-treatment are needed.
Q: How does anammox save cost? A: It converts ammonium directly with nitrite to N₂, so it needs no organic carbon and roughly 60% less aeration than nitrification–denitrification.
Q: Can heavy metals be biodegraded? A: No — elements cannot be destroyed; microbes only change their oxidation state, mobility or solubility, so remediation means biosorption, bioprecipitation or reduction to a less toxic form.
Q: What are biostimulation and bioaugmentation? A: Biostimulation adds nutrients or electron acceptors to boost native degraders; bioaugmentation introduces specialised strains or consortia when natives are absent or too few.
Q: Why do protozoa in an aeration tank indicate good performance? A: Stalked ciliates appear only at long sludge age with adequate DO and low soluble BOD, and their grazing clarifies the effluent.
Q: What is the great plate count anomaly and how is it overcome? A: Under 1% of environmental microbes grow on standard media; culture-independent tools — 16S rRNA amplicon sequencing, metagenomics, qPCR, FISH — profile the rest.
Q: Which Indian standard and Act govern drinking water and effluent discharge? A: IS 10500:2012 specifies drinking water quality (coliforms absent in 100 mL); the Water Act, 1974 and the Environment (Protection) Act, 1986 with its Rules set effluent standards enforced by CPCB and the State Boards.
Q: Why does sludge rise in a secondary clarifier? A: Nitrate carried into the clarifier is denitrified, and the nitrogen gas bubbles lift sludge flocs to the surface; it is cured by reducing clarifier detention or adding an anoxic zone.

Common mistakes

Treating BOD and COD as interchangeable, or quoting BOD₃ at 27 °C figures against BOD₅ at 20 °C standards without conversion.
Forgetting the dilution factor in BOD, or accepting a bottle where depletion is outside 2–6 mg/L or final DO is below 1 mg/L.
Reporting MPN as "cfu/mL" — MPN is a statistical estimate per 100 mL, not a direct colony count.
Saying nitrification removes nitrogen; it only converts ammonia to nitrate — removal needs denitrification or anammox.
Writing that a coliform limit of "10 per 100 mL is permissible" for drinking water; IS 10500:2012 allows none, with no relaxation.
Mixing up *Nitrosomonas* and *Nitrobacter* steps, or calling nitrifiers heterotrophs.
Claiming bioremediation degrades heavy metals, or that phytoremediation is fast — it is cheap but slow and limited to the root zone.