Which sequence correctly represents nitrification followed by its organisms?

NO₃⁻ → N₂ by *Nitrosomonas*, then N₂ → NH₃ by *Nitrobacter*

N₂ → NH₃ by *Nitrobacter*, then NH₃ → NO₃⁻ by *Nitrosomonas*

NO₃⁻ → NO₂⁻ by *Nitrobacter*, then NO₂⁻ → NH₄⁺ by *Nitrosomonas*

Leghaemoglobin in a legume root nodule is essential because it:

keeps free oxygen low enough to protect nitrogenase while still supplying bacteroid respiration

carries carbon dioxide from the bacteroids up to the leaves so the plant can use it for photosynthesis

keeps free oxygen low enough to protect nitrogenase while still supplying bacteroid respiration

directly reduces atmospheric dinitrogen to ammonia in place of nitrogenase

stores nitrate in the nodule for the plant to use during the dry season

A researcher sequences the 16S rRNA gene amplified from total soil DNA, without culturing anything. This approach is best described as:

Amplicon metagenomics, revealing which taxa are present and their proportions

Metatranscriptomics, which sequences community RNA to reveal which genes are being actively expressed

Pure-culture isolation, revealing the physiology of each strain

Amplicon metagenomics, revealing which taxa are present and their proportions

Proteomics, revealing the full set of proteins the community makes

After a coastal oil spill, engineers add nitrogen and phosphorus fertiliser to the beach but no new bacteria. The spilled oil degrades much faster. This is an example of:

Biostimulation, feeding the native oil-degrading microbes a limiting nutrient

Bioaugmentation, introducing specially selected pollutant-degrading bacterial strains into the site

Ex situ landfarming, spreading excavated soil to aerate it

Phytoremediation, using planted vegetation to extract the contaminant

Biostimulation, feeding the native oil-degrading microbes a limiting nutrient

Green and purple sulphur bacteria differ from plants in that their photosynthesis:

uses H₂S as the electron donor and deposits sulphur instead of releasing O₂

uses water as the electron donor and releases oxygen as plants do

uses H₂S as the electron donor and deposits sulphur instead of releasing O₂

fixes nitrogen rather than carbon during the light reactions

requires no light because it is entirely chemolithotrophic

In anaerobic sludge, fermenting bacteria grow well only when methanogens are present to consume the hydrogen they release. Removing the methanogens stalls the fermenters. This dependence is best called:

Syntrophy through interspecies hydrogen transfer

Competition between the two groups for a shared limiting nutrient

Amensalism by an inhibitory metabolic product

Syntrophy through interspecies hydrogen transfer

Predation of the fermenters by the methanogens

Arbuscular mycorrhizae chiefly benefit their host plant by:

improving uptake of phosphate and water through an extended hyphal network

fixing atmospheric nitrogen directly inside the root cortex and passing ammonia to the plant

improving uptake of phosphate and water through an extended hyphal network

photosynthesising extra sugars and donating them to the root

digesting soil nematodes and releasing their nitrogen to the plant

Which of the following statements about microbial interactions are correct?

In commensalism one partner benefits and the other is essentially unaffected

In amensalism one partner is harmed, often by an inhibitory product, while the other is unaffected

In commensalism one partner benefits and the other is essentially unaffected

In amensalism one partner is harmed, often by an inhibitory product, while the other is unaffected

In competition one partner always benefits at the expense of the other

Which of the following statements about biological nitrogen fixation are correct?

It is carried out only by certain prokaryotes, never by plants or animals alone

The enzyme nitrogenase is irreversibly inhibited by oxygen

Cyanobacteria such as *Anabaena* fix nitrogen in specialised heterocysts

It is carried out only by certain prokaryotes, never by plants or animals alone

The enzyme nitrogenase is irreversibly inhibited by oxygen

Cyanobacteria such as *Anabaena* fix nitrogen in specialised heterocysts

Fixation oxidises N₂ to nitrate using atmospheric oxygen

Which statements about culture-independent study of microbial communities are correct?

Typically under 1% of environmental microbes grow readily on standard laboratory media

Shotgun metagenomics can recover functional genes and near-complete genomes

Metatranscriptomics sequences community RNA to show which genes are expressed

Typically under 1% of environmental microbes grow readily on standard laboratory media

Shotgun metagenomics can recover functional genes and near-complete genomes

Metatranscriptomics sequences community RNA to show which genes are expressed

Unculturable microbes are those that have been confirmed dead in the sample

Microbial Ecology: Interactions, Biogeochemical Cycles and Community Genomics

This chapter answers Section XL3.10 of XL3 Microbiology, one of the five optional sections of the Life Sciences (XL) paper: "Microbial Ecology — Microbial interactions; Carbon, sulphur and nitrogen cycles; Soil microorganisms associated with vascular plants; Bioremediation; Uncultivable microorganisms; Basic concept of metagenomics and metatranscriptomics." It teaches how microbes interact in communities, how they drive the carbon, sulphur and nitrogen cycles, the microbes of the rhizosphere and root symbioses, how microbes clean up pollution, why most microbes resist culture, and how metagenomics and metatranscriptomics study them without culturing. The numericals are a nitrogen budget, sequencing coverage depth, Simpson's index of diversity, and the ATP cost of nitrogen fixation.

1. Microbial interactions

Microbes rarely live alone; they form communities whose members interact. Interactions are named by their sign for each partner. In mutualism both partners benefit and the association is obligate — the lichen (a fungus with an alga or cyanobacterium) and the rumen microbes that digest cellulose for the ruminant are classic cases. Protocooperation (synergism) also benefits both but is not obligate. In commensalism one benefits and the other is unaffected, as when one organism's waste is another's substrate. Amensalism (antagonism) harms one partner while the other is unaffected, typically through an inhibitory product such as an antibiotic or an acid. Competition harms both as they vie for a limiting nutrient, and the better competitor may exclude the other. Parasitism and predation harm one partner outright — Bdellovibrio, a bacterium that invades and consumes other Gram-negative bacteria, and protozoa that graze bacteria, are microbial examples.

🎯 Syntrophy: feeding across species

A special mutualism is syntrophy, where one species can grow on a substrate only if a partner removes its product. In anaerobic digestion, fermenting bacteria release hydrogen that is thermodynamically self-limiting; methanogenic archaea consume that hydrogen (interspecies hydrogen transfer), keeping its partial pressure low so fermentation can proceed. Neither partner can do the job alone, which is why so many environmental processes are run by consortia rather than single species.

2. The carbon and sulphur cycles

In the carbon cycle, autotrophs fix CO₂ into organic matter by photosynthesis or chemosynthesis, and all organisms return CO₂ by respiration; decomposer microbes mineralise dead organic carbon back to CO₂. Under anaerobic conditions the cycle takes a methane loop: methanogens (archaea) reduce CO₂ or acetate to methane (CH₄), and methanotrophs (bacteria) oxidise CH₄ back to CO₂ where oxygen is available. Microbes thus govern both the release of greenhouse gases and the turnover of soil organic matter.

Key transformations of the sulphur cycle
Process	Transformation	Organisms
Assimilatory sulphate reduction	SO₄²⁻ → organic sulphur (e.g. cysteine)	Plants, fungi, many bacteria
Dissimilatory sulphate reduction	SO₄²⁻ → H₂S (anaerobic respiration)	Desulfovibrio and other sulphate reducers
Sulphur oxidation	H₂S or S⁰ → SO₄²⁻	Chemolithotrophs (Thiobacillus); photosynthetic sulphur bacteria
Desulfurylation	Organic sulphur → H₂S	Decomposer microbes

The two cycles are coupled in sediments and wetlands: sulphate reducers oxidise organic carbon using sulphate as the electron acceptor, producing H₂S that colours and smells of anoxic mud, while sulphur oxidisers reclaim that H₂S at the oxic interface. Acid mine drainage is the same chemistry turned harmful — chemolithotrophic oxidation of sulphide minerals generates sulphuric acid. These transformations show why a single element's cycle is really a set of linked redox reactions, each run by a guild of microbes specialised for one step.

ℹ️ H₂S as an electron donor

Anoxygenic photosynthetic green and purple sulphur bacteria use H₂S instead of H₂O as the electron donor for photosynthesis, depositing elemental sulphur rather than releasing oxygen. This links the sulphur and carbon cycles and is a reminder that photosynthesis is not always oxygenic.

3. The nitrogen cycle

Nitrogen is abundant as inert N₂ gas but scarce in usable form, so the microbial nitrogen cycle largely controls biological productivity. Its steps are: nitrogen fixation (N₂ → NH₃), carried out only by prokaryotes using the oxygen-sensitive enzyme nitrogenase — free-living fixers include aerobic Azotobacter, anaerobic Clostridium and cyanobacteria such as Anabaena (in specialised heterocysts), and symbiotic fixers include Rhizobium in legume nodules and Frankia in actinorhizal plants; ammonification (organic N → NH₄⁺) by decomposers; nitrification (NH₄⁺ → NO₂⁻ by Nitrosomonas, then NO₂⁻ → NO₃⁻ by Nitrobacter), an aerobic chemolithotrophic process; and denitrification (NO₃⁻ → N₂) by facultative anaerobes such as Pseudomonas, which returns nitrogen to the atmosphere and represents a loss of soil fertility.

Two further reductive steps complete the picture. Assimilatory nitrate reduction lets plants and microbes convert NO₃⁻ back to NH₃ for building amino acids, so nitrate taken up is not wasted. The cycle is therefore a balance between processes that make nitrogen available (fixation, ammonification, nitrification feeding nitrate uptake) and those that remove it (denitrification, anammox, and leaching of the very soluble nitrate below the root zone). Because nitrate is mobile and ammonium is held on clay, the form in which nitrogen sits in soil strongly shapes how much is lost.

🧠 Nitrogenase, its cost, and anammox

Fixing dinitrogen is expensive: the simplified stoichiometry is N₂ + 8 H⁺ + 8 e⁻ + 16 ATP → 2 NH₃ + H₂ + 16 ADP + 16 Pᵢ, so 16 ATP are spent per N₂ (8 per ammonia). Nitrogenase is irreversibly poisoned by O₂, which is why aerobes protect it — by heterocysts in cyanobacteria and by leghaemoglobin mopping up O₂ in legume nodules. A separate anaerobic shortcut, anammox (NH₄⁺ + NO₂⁻ → N₂), carried out by planctomycete bacteria, returns a large share of oceanic nitrogen to N₂.

4. Soil microorganisms associated with vascular plants

The rhizosphere is the thin zone of soil around a root, enriched by root exudates (sugars, amino acids, organic acids) that support far more microbes than bulk soil — the rhizosphere-to-soil (R:S) ratio of microbial numbers is often 10–100. Several associations are agriculturally central. Mycorrhizae are root–fungus symbioses that greatly extend the absorbing surface for phosphate and water in exchange for plant sugars: arbuscular (endo-)mycorrhizae (Glomeromycota) penetrate cortical cells to form arbuscules and occur in most crops, while ectomycorrhizae form a sheath and Hartig net around the roots of many trees.

The legume–Rhizobium symbiosis is the best-studied: Nod factors trigger root-hair curling and nodule formation, the bacteria differentiate into bacteroids, and plant leghaemoglobin holds free O₂ low enough to protect nitrogenase while still feeding bacteroid respiration. Plant growth-promoting rhizobacteria (PGPR) such as Azospirillum, Pseudomonas and Bacillus aid plants without nodulating them — fixing some nitrogen, solubilising phosphate, producing phytohormones and iron-scavenging siderophores, and suppressing pathogens (biocontrol). The water fern Azolla, with its cyanobacterial symbiont, is a traditional green manure for rice.

5. Bioremediation

Bioremediation uses microbes (or their enzymes) to degrade or detoxify pollutants into harmless products such as CO₂, water and biomass. It is cheaper and less disruptive than digging out and incinerating contaminated soil, but it is slower and limited by what the microbes can reach and tolerate. Organic pollutants — petroleum hydrocarbons, solvents, pesticides, polycyclic aromatics — are broken down by bacteria such as Pseudomonas and Alcanivorax (an oil-degrading specialist); some compounds are attacked only by co-metabolism, where the microbe transforms the pollutant while growing on something else. Toxic metals are not destroyed but can be immobilised or changed in valency — microbial reduction of soluble, toxic chromium(VI) to insoluble chromium(III), or of mercuric ion by the mer system, lowers their hazard.

Success depends on three things the microbes cannot change by themselves: oxygen (aerobic degradation of hydrocarbons is far faster than anaerobic, so aeration helps), nutrients (the carbon-rich pollutant must be balanced with nitrogen and phosphorus), and bioavailability (a pollutant bound tightly to soil or sealed inside an oily droplet is out of reach, which is why surfactants and tilling are used). Recalcitrant compounds such as highly chlorinated solvents resist attack and may need an anaerobic dechlorinating community first, followed by aerobic polishing.

In situ methods treat the pollution in place: biostimulation (adding nitrogen, phosphorus or oxygen to feed native microbes, as in fertilising an oil spill), bioaugmentation (adding selected strains), bioventing and natural attenuation.
Ex situ methods remove the material first: landfarming, composting, biopiles and slurry bioreactors, where conditions can be controlled more tightly.

6. Unculturable microbes, metagenomics and metatranscriptomics

The great plate count anomaly is the finding that far fewer microbes grow on laboratory media than are seen under the microscope — typically less than 1% of an environmental community is readily culturable. The rest are not dead but unculturable under standard conditions: they may need partners (syntrophy), unknown growth factors, very slow growth, or specific chemistry, and some enter a viable-but-nonculturable (VBNC) state. Approaches that recover a few of them include dilution-to-extinction, diffusion chambers and the iChip (growth in situ), and co-culture; but the majority are studied by culture-independent molecular methods.

💡 Potential versus activity

Think of metagenomics as reading a factory's full list of machines (the genes it could run) and metatranscriptomics as watching which machines are switched on right now (the genes being transcribed). A community can carry a denitrification gene yet express it only when oxygen runs low, so DNA tells you the capability and RNA tells you the realised process at the moment of sampling.

Metagenomics sequences the total DNA extracted directly from an environment, bypassing culture altogether. Amplicon metagenomics sequences one marker gene — usually the 16S rRNA gene for prokaryotes — to answer who is there and in what proportions; shotgun metagenomics sequences all the DNA to answer what they can do, recovering functional genes and even near-complete genomes (metagenome-assembled genomes). Metatranscriptomics sequences the community's total RNA (mRNA) to show which genes are actually being expressed in situ — the difference between genetic potential and realised activity. The reads are grouped into OTUs or exact sequence variants and summarised by diversity indices; the depth of sequencing (coverage) determines how much of the rare community is seen.

Key takeaways

Microbial interactions are named by their sign for each partner: mutualism (both benefit, obligate), protocooperation, commensalism, amensalism, competition, parasitism and predation; syntrophy (interspecies hydrogen transfer) lets consortia do what no single species can.
Microbes drive the carbon cycle (fixation, respiration, methanogenesis and methanotrophy) and the sulphur cycle (assimilatory and dissimilatory sulphate reduction, sulphur oxidation, desulfurylation); some photosynthesisers use H₂S rather than H₂O.
The nitrogen cycle is fixation (nitrogenase, 16 ATP per N₂, O₂-sensitive), ammonification, nitrification (Nitrosomonas then Nitrobacter, aerobic) and denitrification (to N₂, anaerobic); anammox is an anaerobic NH₄⁺ + NO₂⁻ → N₂ shortcut.
The rhizosphere teems with microbes fed by root exudates; mycorrhizae (arbuscular and ecto-) boost phosphate and water uptake, Rhizobium nodulates legumes (leghaemoglobin protects nitrogenase), and PGPR promote growth without nodules.
Bioremediation degrades organic pollutants (hydrocarbons by Pseudomonas, Alcanivorax; sometimes by co-metabolism) and immobilises or changes the valency of metals; in situ methods (biostimulation, bioaugmentation) treat in place, ex situ methods remove first.
Under 1% of environmental microbes are culturable (the great plate count anomaly); culture-independent metagenomics (16S amplicon for who, shotgun for what) and metatranscriptomics (RNA for which genes are active) reveal the rest.

Practice questions (16)

Attempt each one before opening the answer. Every explanation names the tempting wrong option as well as the right one, because that is where marks are lost.

In a lichen, a fungus and a photosynthetic partner live together and both benefit, neither surviving the association's loss. This interaction is:
1. Commensalism
2. Amensalism
3. Parasitism
4. Mutualism
Show answer
Answer: D — Mutualism
Both partners benefit and the relationship is obligate, which is mutualism. Commensalism benefits only one and leaves the other unaffected; amensalism harms one; parasitism harms one partner for the gain of the other — none of which fits a relationship good for both.
Which sequence correctly represents nitrification followed by its organisms?
1. NO₃⁻ → N₂ by Nitrosomonas, then N₂ → NH₃ by Nitrobacter
2. N₂ → NH₃ by Nitrobacter, then NH₃ → NO₃⁻ by Nitrosomonas
3. NH₄⁺ → NO₂⁻ by Nitrosomonas, then NO₂⁻ → NO₃⁻ by Nitrobacter
4. NO₃⁻ → NO₂⁻ by Nitrobacter, then NO₂⁻ → NH₄⁺ by Nitrosomonas
Show answer
Answer: C — NH₄⁺ → NO₂⁻ by *Nitrosomonas*, then NO₂⁻ → NO₃⁻ by *Nitrobacter*
Nitrification is the aerobic oxidation of ammonium to nitrate in two steps: Nitrosomonas oxidises NH₄⁺ to nitrite, then Nitrobacter oxidises nitrite to nitrate. The reductive sequence NO₃⁻ → N₂ is denitrification, and N₂ → NH₃ is fixation; swapping oxidation for reduction, or the two genera, is the usual error.
Leghaemoglobin in a legume root nodule is essential because it:
1. carries carbon dioxide from the bacteroids up to the leaves so the plant can use it for photosynthesis
2. keeps free oxygen low enough to protect nitrogenase while still supplying bacteroid respiration
3. directly reduces atmospheric dinitrogen to ammonia in place of nitrogenase
4. stores nitrate in the nodule for the plant to use during the dry season
Show answer
Answer: B — keeps free oxygen low enough to protect nitrogenase while still supplying bacteroid respiration
Nitrogenase is irreversibly inactivated by O₂, yet the bacteroids need O₂ to respire; leghaemoglobin binds O₂ and buffers its free concentration low, protecting the enzyme while delivering oxygen for respiration. It does not itself fix nitrogen (that is nitrogenase), does not transport CO₂ to leaves, and does not store nitrate.
A researcher sequences the 16S rRNA gene amplified from total soil DNA, without culturing anything. This approach is best described as:
1. Metatranscriptomics, which sequences community RNA to reveal which genes are being actively expressed
2. Pure-culture isolation, revealing the physiology of each strain
3. Amplicon metagenomics, revealing which taxa are present and their proportions
4. Proteomics, revealing the full set of proteins the community makes
Show answer
Answer: C — Amplicon metagenomics, revealing which taxa are present and their proportions
Sequencing an amplified marker gene (16S rRNA) from community DNA is amplicon metagenomics; the 16S gene answers 'who is there' and in what relative abundance. Metatranscriptomics sequences RNA to show expression, culture isolation needs growth (ruled out here), and proteomics measures proteins, not the 16S gene.
After a coastal oil spill, engineers add nitrogen and phosphorus fertiliser to the beach but no new bacteria. The spilled oil degrades much faster. This is an example of:
1. Bioaugmentation, introducing specially selected pollutant-degrading bacterial strains into the site
2. Ex situ landfarming, spreading excavated soil to aerate it
3. Phytoremediation, using planted vegetation to extract the contaminant
4. Biostimulation, feeding the native oil-degrading microbes a limiting nutrient
Show answer
Answer: D — Biostimulation, feeding the native oil-degrading microbes a limiting nutrient
Adding a limiting nutrient (here N and P) to stimulate the pollutant-degrading microbes already present is biostimulation; hydrocarbon-rich spills are usually carbon-rich but nitrogen- and phosphorus-poor, so fertilising removes the bottleneck. Bioaugmentation would mean adding microbes, which was explicitly not done; landfarming is ex situ (soil was not excavated), and phytoremediation uses plants.
Green and purple sulphur bacteria differ from plants in that their photosynthesis:
1. uses water as the electron donor and releases oxygen as plants do
2. uses H₂S as the electron donor and deposits sulphur instead of releasing O₂
3. fixes nitrogen rather than carbon during the light reactions
4. requires no light because it is entirely chemolithotrophic
Show answer
Answer: B — uses H₂S as the electron donor and deposits sulphur instead of releasing O₂
Anoxygenic sulphur bacteria use H₂S (not H₂O) as the electron donor, so they deposit elemental sulphur and release no oxygen — the defining contrast with oxygenic plant photosynthesis. They do not split water, photosynthesis fixes carbon not nitrogen, and these are phototrophs that do need light.
In anaerobic sludge, fermenting bacteria grow well only when methanogens are present to consume the hydrogen they release. Removing the methanogens stalls the fermenters. This dependence is best called:
1. Competition between the two groups for a shared limiting nutrient
2. Amensalism by an inhibitory metabolic product
3. Syntrophy through interspecies hydrogen transfer
4. Predation of the fermenters by the methanogens
Show answer
Answer: C — Syntrophy through interspecies hydrogen transfer
The fermenters' hydrogen-producing reactions are thermodynamically favourable only when hydrogen is kept low; methanogens do exactly that by consuming it, so each partner depends on the other — syntrophy via interspecies hydrogen transfer. It is not competition (they are not vying for one resource), not amensalism (the partner helps, not harms), and not predation (the methanogens do not eat the fermenters).
Arbuscular mycorrhizae chiefly benefit their host plant by:
1. fixing atmospheric nitrogen directly inside the root cortex and passing ammonia to the plant
2. improving uptake of phosphate and water through an extended hyphal network
3. photosynthesising extra sugars and donating them to the root
4. digesting soil nematodes and releasing their nitrogen to the plant
Show answer
Answer: B — improving uptake of phosphate and water through an extended hyphal network
Mycorrhizal hyphae extend far beyond the root's own reach and greatly improve uptake of poorly mobile nutrients, above all phosphate, plus water, in exchange for plant sugars. Mycorrhizal fungi do not fix nitrogen (that is Rhizobium/Frankia), are heterotrophs that take sugar rather than make it, and are not nematode predators.
Which process returns fixed nitrogen from soil back to atmospheric N₂, representing a loss of soil fertility?
1. Denitrification
2. Nitrification
3. Ammonification
4. Nitrogen fixation
Show answer
Answer: A — Denitrification
Denitrification reduces nitrate to N₂ (via NO₂⁻, NO and N₂O) under anaerobic conditions, returning nitrogen to the air and depleting soil nitrate. Nitrification oxidises ammonium to nitrate, ammonification releases ammonium from organic matter, and fixation adds nitrogen — the opposite of a loss.
Which of the following statements about microbial interactions are correct?
1. In mutualism both partners benefit
2. In commensalism one partner benefits and the other is essentially unaffected
3. In amensalism one partner is harmed, often by an inhibitory product, while the other is unaffected
4. In competition one partner always benefits at the expense of the other
Show answer
Answer: A — In mutualism both partners benefit; B — In commensalism one partner benefits and the other is essentially unaffected; C — In amensalism one partner is harmed, often by an inhibitory product, while the other is unaffected
Mutualism (+/+), commensalism (+/0) and amensalism (−/0) are correctly described. Competition harms both partners (−/−) as they draw on the same limiting resource; it is not a one-sided gain, so that statement is wrong — the one-sided descriptions fit parasitism or predation instead.
Which of the following statements about biological nitrogen fixation are correct?
1. It is carried out only by certain prokaryotes, never by plants or animals alone
2. The enzyme nitrogenase is irreversibly inhibited by oxygen
3. Cyanobacteria such as Anabaena fix nitrogen in specialised heterocysts
4. Fixation oxidises N₂ to nitrate using atmospheric oxygen
Show answer
Answer: A — It is carried out only by certain prokaryotes, never by plants or animals alone; B — The enzyme nitrogenase is irreversibly inhibited by oxygen; C — Cyanobacteria such as *Anabaena* fix nitrogen in specialised heterocysts
Only prokaryotes possess nitrogenase (plants fix nitrogen solely through their prokaryotic symbionts), the enzyme is O₂-sensitive, and Anabaena isolates it in heterocysts. Fixation is a reduction of N₂ to ammonia, not an oxidation to nitrate, and it does not use oxygen — the claim that fixation oxidises N₂ to nitrate inverts the chemistry.
Which statements about culture-independent study of microbial communities are correct?
1. Typically under 1% of environmental microbes grow readily on standard laboratory media
2. Shotgun metagenomics can recover functional genes and near-complete genomes
3. Metatranscriptomics sequences community RNA to show which genes are expressed
4. Unculturable microbes are those that have been confirmed dead in the sample
Show answer
Answer: A — Typically under 1% of environmental microbes grow readily on standard laboratory media; B — Shotgun metagenomics can recover functional genes and near-complete genomes; C — Metatranscriptomics sequences community RNA to show which genes are expressed
The great plate count anomaly means under ~1% are readily cultured, shotgun metagenomics reveals functions and assembled genomes, and metatranscriptomics reads RNA for expression. 'Unculturable' does not mean dead: such cells are usually alive but will not grow under the conditions tried, which is exactly why molecular methods are needed.
A legume crop fixes 150 kg of nitrogen per hectare in a season, while the harvested grain and straw remove 200 kg of nitrogen per hectare. The net nitrogen that must be supplied from other sources to balance the budget is ____ kg per hectare.
Numerical answer — type the value.
Show answer
Answer: 50
Net requirement = removal − fixation = 200 − 150 = 50 kg N per hectare. Fixation offsets part of what the crop exports, but a 50 kg deficit per hectare remains and must come from residues, manure or fertiliser.
In a shotgun metagenome, 1,000,000 reads of 150 bp each are mapped to a reference genome of 5,000,000 bp. Using coverage = (number of reads × read length) / genome size, the average coverage depth is ____ ×.
Numerical answer — type the value.
Show answer
Answer: 30
Coverage = (N × L) / G = (1,000,000 × 150) / 5,000,000 = 150,000,000 / 5,000,000 = 30×. On average each base of the genome is read 30 times, which is ample for confident variant and gene calling.
A soil sample holds three bacterial taxa in relative proportions 0.5, 0.3 and 0.2. Using Simpson's index of diversity, 1 − Σ(pᵢ²), the diversity is ____ (to two decimal places).
Numerical answer — type the value.
Show answer
Answer: 0.62
Σ(pᵢ²) = 0.5² + 0.3² + 0.2² = 0.25 + 0.09 + 0.04 = 0.38, so Simpson's index of diversity = 1 − 0.38 = 0.62. It is the probability that two individuals drawn at random belong to different taxa; a higher value means greater diversity.
In the simplified nitrogenase reaction N₂ + 8 H⁺ + 8 e⁻ + 16 ATP → 2 NH₃ + H₂ + 16 ADP + 16 Pᵢ, the number of ATP molecules consumed to fix one molecule of N₂ is ____.
Numerical answer — type the value.
Show answer
Answer: 16
The stoichiometry shows 16 ATP are hydrolysed per N₂ reduced, i.e. 8 ATP per ammonia formed. This large energy cost is why nitrogen fixation is tightly regulated and switched off when fixed nitrogen is already available.