6.1: Introduction to Oxygen Requirements - Biology

6.1: Introduction to Oxygen Requirements - Biology

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Learning Outcomes

  • Recognize the effects of Oxygen on bacteria
  • Explain the various oxygen requirements of the microbes, observe and interpret the growth of microbes in thioglycollate agar deep media
  • Discuss methods of culturing anaerobic bacteria

Environmental Requirements: Oxygen requirements

How does Oxygen affect bacterial growth?

Bacteria can differ dramatically in their ability to utilize oxygen (O2). Under aerobic conditions, oxygen acts as the final electron acceptor for the electron transport chain located in the plasma membrane of prokaryotes. Bacteria use this process to generate ATP, the energy source for most cellular processes. In the absence of oxygen (O2), some bacteria can use alternative metabolic pathways including anaerobic respiration and/or fermentation. During anaerobic respiration, other alternative molecules are used as the final electron acceptor for the electron transport chain such as nitrate (NO3), sulfate (SO4), and carbonate (CO3).

Bacteria and many microorganisms are very sensitive to oxygen concentrations. Some will only grow in its presence and are called obligate aerobes. Facultative aerobes will grow either aerobically or in the absence of oxygen (anaerobic conditions), but they generally do better with oxygen. Aerotolerant anaerobes don't require oxygen, but can grow in its presence, while strict obligate anaerobes cannot use oxygen and cannot grow or survive in its presence. Microaerophiles use oxygen, but at lower concentrations than atmospheric oxygen levels (which is ~20%).

One can determine a bacterium's oxygen requirements by cultivating them in a special medium called thioglycollate agar tubes. The bottom of the tube of medium is kept anaerobic by cystine and thioglycollic acid, which chemically react with, and tie up any oxygen that diffuses in. Any un-reacted oxygen in the tube will be indicated by resazurin, a dye that turns pink in the presence of oxygen. It is common for the top centimeter or so to be pink. One can inoculate a thioglycollate tube with your bacterium and observe where the bacterium grows in the tube to determine its oxygen requirements (see Image 1)

Image 1: Microbial oxygen requirements determined using thioglycollate agar tubes. Green dots represent bacterial colonies within in the agar or on its surface. The surface of the agar tube is directly exposed to atmospheric oxygen, and will be aerobic. The oxygen content of the thioglycollate medium decreases with depth until the medium becomes anaerobic towards the bottom of the tube.

Cultivation of Anaerobes

The cultivation of anaerobes can be done in anaerobic chamber (image 2). This is a special chamber where you can work with and cultivate strict obligate anaerobes without exposing them to oxygen. Anaerobic chambers contain a hydrogen (H2) gas mixture that is circulated through a heated palladium catalyst to remove oxygen (O2) by forming water (H2O). Anaerobic chambers use a gas mixture of H2 and nitrogen gas (N2) (5/95%) or N2/carbon dioxide (CO2)/H2 (85/10/5 %) to remove oxygen. An airlock is used to reduce O2 levels prior to the transfer of samples in and out of the chamber.

Another way of culturing bacteria anaerobically on plates is to use a GasPak anaerobic system. In these systems, hydrogen and carbon dioxide are generated by a GasPak envelope after the addition of water. A palladium catalyst in the chamber of the GasPak system catalyzes the formation of water from hydrogen and oxygen, thereby removing oxygen from the sealed chamber (image 3 and 4). These systems are compact, easy to use, and less expensive than an anaerobic chamber. They come in jars (image 3) or in a box format (image 4).

Image 2:Anaerobic chamber.

Image 3: GasPak system jars Image 4: GasPak system boxes.

Watch Video 1: Basics of thioglycollate media

Watch Video 1: explanation on how thioglycollate media works and examples. (9:33) URL:

Watch Video 2: how to prepare an anaerobic jar

Watch Video 2: how to set up an anaerobic jar. The process is similar for an anaerobic box. (3:03) URL:

Reactive oxygen species

Reactive oxygen species (ROS) are highly reactive chemical molecules formed due to the electron receptivity of O2. Examples of ROS include peroxides, superoxide, hydroxyl radical, singlet oxygen, [3] and alpha-oxygen.

The reduction of molecular oxygen (O2) produces superoxide ( • O −
2 ), which is the precursor of most other reactive oxygen species: [4]

Hydrogen peroxide in turn may be partially reduced, thus forming hydroxide ions and hydroxyl radicals ( • OH), or fully reduced to water: [4]

In a biological context, ROS are formed as a natural byproduct of the normal aerobic metabolism of oxygen and have important roles in cell signaling and homeostasis. [5] [6] ROS are intrinsic to cellular functioning, and are present at low and stationary levels in normal cells. In vegetables, ROS are involved in metabolic processes related to photoprotection and tolerance to different types of stress. [7] However, ROS can cause irreversible damage to DNA as they oxidize and modify some cellular components and prevent them from performing their original functions. This suggests that ROS has a dual role whether they will act as harmful, protective or signaling factors depends on the balance between ROS production and disposal at the right time and place. [8] In other words, oxygen toxicity can arise both from uncontrolled production and from the inefficient elimination of ROS by the antioxidant system. During times of environmental stress (e.g., UV or heat exposure), ROS levels can increase dramatically. [5] This may result in significant damage to cell structures. Cumulatively, this is known as oxidative stress. The production of ROS is strongly influenced by stress factor responses in plants, these factors that increase ROS production include drought, salinity, chilling, defense of pathogens, nutrient deficiency, metal toxicity and UV-B radiation. ROS are also generated by exogenous sources such as ionizing radiation [9] generating irreversible effects in the development of tissues in both animals and plants. [10]

Dissolved Oxygen and Water

Dissolved oxygen (DO) is a measure of how much oxygen is dissolved in the water - the amount of oxygen available to living aquatic organisms. The amount of dissolved oxygen in a stream or lake can tell us a lot about its water quality.

USGS scientist is measuring various water-quality conditions in Holes Creek at Huffman Park in Kettering, Ohio.

The USGS has been measuring water for decades. Some measurements, such as temperature, pH, and specific conductance are taken almost every time water is sampled and investigated, no matter where in the U.S. the water is being studied. Another common measurement often taken is dissolved oxygen (DO), which is a measure of how much oxygen is dissolved in the water - DO can tell us a lot about water quality.

Dissolved Oxygen and Water

Although water molecules contain an oxygen atom, this oxygen is not what is needed by aquatic organisms living in natural waters. A small amount of oxygen, up to about ten molecules of oxygen per million of water, is actually dissolved in water. Oxygen enters a stream mainly from the atmosphere and, in areas where groundwater discharge into streams is a large portion of streamflow, from groundwater discharge. This dissolved oxygen is breathed by fish and zooplankton and is needed by them to survive.

Dissolved oxygen and water quality

A eutrophic lake where dissolved-oxygen concentrations are low. Algal blooms can occur under such conditions.

Rapidly moving water, such as in a mountain stream or large river, tends to contain a lot of dissolved oxygen, whereas stagnant water contains less. Bacteria in water can consume oxygen as organic matter decays. Thus, excess organic material in lakes and rivers can cause eutrophic conditions, which is an oxygen-deficient situation that can cause a water body to "die." Aquatic life can have a hard time in stagnant water that has a lot of rotting, organic material in it, especially in summer (the concentration of dissolved oxygen is inversely related to water temperature), when dissolved-oxygen levels are at a seasonal low. Water near the surface of the lake– the epilimnion– is too warm for them, while water near the bottom–the hypolimnion– has too little oxygen. Conditions may become especially serious during a period of hot, calm weather, resulting in the loss of many fish. You may have heard about summertime fish kills in local lakes that likely result from this problem.

Dissolved oxygen, temperature, and aquatic life

Water temperture affects dissolved-oxygen concentrations in a river or water body.

As the chart shows, the concentration of dissolved oxygen in surface water is affected by temperature and has both a seasonal and a daily cycle. Cold water can hold more dissolved oxygen than warm water. In winter and early spring, when the water temperature is low, the dissolved oxygen concentration is high. In summer and fall, when the water temperature is high, the dissolved-oxygen concentration is often lower.

Dissolved oxygen in surface water is used by all forms of aquatic life therefore, this constituent typically is measured to assess the "health" of lakes and streams. Oxygen enters a stream from the atmosphere and from groundwater discharge. The contribution of oxygen from groundwater discharge is significant, however, only in areas where groundwater is a large component of streamflow, such as in areas of glacial deposits. Photosynthesis is the primary process affecting the dissolved-oxygen/temperature relation water clarity and strength and duration of sunlight, in turn, affect the rate of photosynthesis.

Hypoxia and "Dead zones"

You may have heard about a Gulf of Mexico "dead zone" in areas of the Gulf south of Louisiana, where the Mississippi and Atchafalaya Rivers discharge. A dead zone forms seasonally in the northern Gulf of Mexico when subsurface waters become depleted in dissolved oxygen and cannot support most life. The zone forms west of the Mississippi Delta over the continental shelf off Louisiana and sometimes extends off Texas. The oxygen depletion begins in late spring, increases in summer, and ends in the fall.

Dissolved oxygen in bottom waters, measured from June 8 through July 17, 2009, during the annual summer Gulf of Mexico Southeast Area Monitoring and Assessment Program ( SEAMAP ) cruise in the northern Gulf of Mexico. Orange and red colors indicate lower dissolved oxygen concentrations.

The formation of oxygen-depleted subsurface waters has been associated with nutrient-rich (nitrogen and phosphorus) discharge from the Mississippi and Atchafalaya Rivers. Bio-available nutrients in the discharge can stimulate algal blooms, which die and are eaten by bacteria, depleting the oxygen in the subsurface water. The oxygen content of surface waters of normal salinity in the summer is typically more than 8 milligrams per liter (8 mg/L) when oxygen concentrations are less than 2 mg/L, the water is defined as hypoxic (CENR, 2000). The hypoxia kills many organisms that cannot escape, and thus the hypoxic zone is informally known as the “dead zone.”

The hypoxic zone in the northern Gulf of Mexico is in the center of a productive and valuable fishery. The increased frequency and expansion of hypoxic zones have become an important economic and environmental issue to commercial and recreational users of the fishery.

Measuring dissolved oxygen

Multi-parameter monitor used to record water-quality measurements.

Field and lab meters to measure dissolved oxygen have been around for a long time. As this picture shows, modern meters are small and highly electronic. They still use a probe, which is located at the end of the cable. Dissolved oxygen is dependent on temperature (an inverse relation), so the meter must be calibrated properly before each use.

Do you want to test your local water quality?

Water test kits are available from World Water Monitoring Challenge (WWMC), an international education and outreach program that builds public awareness and involvement in protecting water resources around the world. Teachers and water-science enthusiasts: Do you want to be able to perform basic water-quality tests on local waters? WWMC offers inexpensive test kits so you can perform your own tests for temperature, pH, turbidity, and dissolved oxygen.

Do you think you know a lot about water properties?
Take our interactive water-properties true/false quiz and test your water knowledge.

Bioinorganic Fundamentals and Applications: Metals in Natural Living Systems and Metals in Toxicology and Medicine Fundamental Properties of Dioxygen

Dioxygen is a major component of the earth's atmosphere comprising

20% by volume. Molecular oxygen is a ground-state triplet, consisting of two unpaired electrons one in each of the doubly degenerate π* HOMOs. Formally, molecular oxygen has a double bond and is thermodynamically a powerful oxidant. However due to spin conservation, the reaction of O2 with ground-state singlet molecules is kinetically unfavorable and requires the reaction with other ground-state radicals, such as flavins, pterins, or metal ions. As shown in Figure 1 , the one-electron reduction of dioxygen to superoxide is thermodynamically unfavorable. However, the two-electron reduction of dioxygen and the one-electron reduction of superoxide to peroxide are thermodynamically favorable and lead to O–O bond elongation as electrons are added to the antibonding π* orbitals (see Figure 5 15 for the physical properties of dioxygen-containing moieties).

Figure 5 . Physical properties of molecular oxygen and reduced dioxygen moieties.

Adapted from Conry, R. R. Encyclopedia of Inorganic Chemistry, John Wiley and Sons: Hoboken, 2006.

The coordination of O2 to copper in both enzymatic and synthetic systems involves a large degree of electron transfer from the reduced copper center to molecular oxygen in what is most often believed to be an inner-sphere mechanism, i.e., complex formation includes bond formation accompanied by electron transfer. The nature of the copper–dioxygen adduct formed is highly variable and depends on many factors including the ligand/protein type (e.g., S vs. N), resulting coordination number and geometry, the number of copper ions in close proximity, etc. (see Figure 6 ).

Figure 6 . Synthetically derived mono- and dinuclear copper complexes ligated to O2-derived species. Underlined species have been observed in biological systems.

Primary and Secondary Detection Reagents

Both enzyme and macrofluorophore labels can be coupled directly to target-specific affinity reagents (primary detection) or to more generic affinity reagents that form stable complexes with unlabeled primary reagents, usually on the basis of immunorecognition (secondary detection). As indicated schematically in Figure 6.1.1, secondary detection inherently provides some degree of signal amplification, although sometimes at the expense of additional background due to nonspecific binding. These basic concepts of primary and secondary detection apply not only to the signal amplification techniques addressed in the current chapter but also to the dye-labeled affinity reagents described in Antibodies, Avidins and Lectins—Chapter 7.

Primary Detection Reagents

Any easily detectable molecule that binds directly to a specific target is a primary detection reagent. Such reagents are detected by fluorescence, chemiluminescence, absorption or electron diffraction conferred by stably attached labels. The conjugation and crosslinking chemistries used to create these stable attachments are discussed in detail in Fluorophores and Their Amine-Reactive Derivatives—Chapter 1, Thiol-Reactive Probes—Chapter 2 and Crosslinking and Photoactivatable Reagents—Chapter 5. In addition to our fluorophore-labeled anti-dye antibodies (Anti-Dye and Anti-Hapten Antibodies—Section 7.4) and monoclonal antibodies (, many of the Molecular Probes site-selective products can be considered primary detection reagents. These include our fluorescent lectins (Lectins and Other Carbohydrate-Binding Proteins—Section 7.7), nucleic acid stains (Nucleic Acid Detection and Analysis—Chapter 8), protein and glycoprotein stains (Protein Detection on Gels, Blots and Arrays—Section 9.3, Detecting Protein Modifications—Section 9.4), phallotoxins (Probes for Actin—Section 11.1), membrane probes (Probes for Lipids and Membranes—Chapter 13), annexin V conjugates for detecting apoptotic cells (Assays for Apoptosis—Section 15.5) and various drug and toxin analogs (Probes for Neurotransmitter Receptors—Section 16.2, Probes for Ion Channels and Carriers—Section 16.3). These primary detection reagents can typically be detected by fluorescence microscopy, fluorometry or flow cytometry methods.

Secondary Detection Reagents

Although many biomolecules, such as antibodies and lectins, bind selectively to a biological target, they usually need to be chemically modified before they can be detected. Often the biomolecule is conjugated to a fluorescent or chromophoric dye or to a heavy atom complex such as colloidal gold. However, the researcher may wish to avoid the time and expense required for these conjugations, choosing instead to use a more generic secondary detection reagent. Typically, secondary detection reagents recognize a particular class of molecules. For example, labeled goat anti–mouse IgG antibodies can be used to localize a tremendous variety of target-specific mouse monoclonal antibodies. Our extensive secondary antibody offering (Secondary Immunoreagents—Section 7.2) provides a wide selection of labels including our superior Alexa Fluor dye series, phycobiliproteins, Alexa Fluor dye–phycobiliprotein tandem fluorophores, Qdot nanocrystals, biotin and enzyme labels (HRP and alkaline phosphatase). We also offer many options in terms of immunoreactivity, an essential consideration in avoiding confounding cross-reactivity when performing simultaneous secondary immunodetection of two or more targets. Our labeled secondary antibody portfolio contains antibodies against IgG and IgM from several mammalian species, including various isotypes of mouse IgG, as well as antibodies against avian (chicken) IgY. Our Zenon antibody labeling technology (Zenon Technology: Versatile Reagents for Immunolabeling—Section 7.3) uses conjugates of an Fc-specific anti-IgG Fab fragment for the rapid and quantitative labeling of the corresponding mouse, rabbit, goat or human antibody.


Oxygen is used as a medical treatment in both chronic and acute cases, and can be used in hospital, pre-hospital or entirely out of hospital.

Chronic conditions Edit

A common use of supplementary oxygen is in people with chronic obstructive pulmonary disease (COPD), the occurrence of chronic bronchitis or emphysema, a common long-term effect of smoking, who may require additional oxygen to breathe either during a temporary worsening of their condition, or throughout the day and night. It is indicated in people with COPD, with arterial oxygen partial pressure Pa O
2 ≤ 55 mmHg (7.3 kPa) or arterial oxygen saturation Sa O
2 ≤ 88% and has been shown to increase lifespan. [13] [14] [15]

Oxygen is often prescribed for people with breathlessness, in the setting of end-stage cardiac or respiratory failure, advanced cancer or neurodegenerative disease, despite having relatively normal blood oxygen levels. A 2010 trial of 239 subjects found no significant difference in reducing breathlessness between oxygen and air delivered in the same way. [16]

Acute conditions Edit

Oxygen is widely used in emergency medicine, both in hospital and by emergency medical services or those giving advanced first aid.

In the pre-hospital environment, high-flow oxygen is indicated for use in resuscitation, major trauma, anaphylaxis, major bleeding, shock, active convulsions, and hypothermia. [17] [18]

It may also be indicated for any other people where their injury or illness has caused low oxygen levels, although in this case oxygen flow should be moderated to achieve oxygen saturation levels, based on pulse oximetry (with a target level of 94–96% in most, or 88–92% in people with COPD). [17] [8] Excessive use of oxygen in those who are acutely ill however increases the risk of death. [8] In 2018 recommendations within the British Medical Journal were that oxygen should be stopped if saturations are greater than 96% and should not be started if above 90 to 93%. [19] Exceptions were those with carbon monoxide poisoning, cluster headaches, attacks of sickle cell disease, and pneumothorax. [19]

For personal use, high concentration oxygen is used as home therapy to abort cluster headache attacks, due to its vaso-constrictive effects. [20]

People who are receiving oxygen therapy for low oxygen following an acute illness or hospitalization should not routinely have a prescription renewal for continued oxygen therapy without a physician's re-assessment of the person's condition. [21] If the person has recovered from the illness, then the hypoxemia is expected to resolve and additional care would be unnecessary and a waste of resources. [21]

Many EMS protocols indicate that oxygen should not be withheld from anyone, while other protocols are more specific or circumspect. However, there are certain situations in which oxygen therapy is known to have a negative impact on a person's condition. [22]

Oxygen should never be given to a person who has paraquat poisoning unless they have severe respiratory distress or respiratory arrest, as this can increase the toxicity. Paraquat poisoning is rare with about 200 deaths globally from 1958 to 1978. [23] Oxygen therapy is not recommended for people who have pulmonary fibrosis or other lung damage resulting from bleomycin treatment. [24]

High levels of oxygen given to infants cause blindness by promoting overgrowth of new blood vessels in the eye obstructing sight. This is retinopathy of prematurity (ROP).

Oxygen has vasoconstrictive effects on the circulatory system, reducing peripheral circulation and was once thought to potentially increase the effects of stroke. However, when additional oxygen is given to the person, additional oxygen is dissolved in the plasma according to Henry's Law. This allows a compensating change to occur and the dissolved oxygen in plasma supports embarrassed (oxygen-starved) neurons, reduces inflammation and post-stroke cerebral edema. Since 1990, hyperbaric oxygen therapy has been used in the treatments of stroke on a worldwide basis. In rare instances, people receiving hyperbaric oxygen therapy have had seizures. However, because of the aforementioned Henry's Law effect of extra available dissolved oxygen to neurons, there is usually no negative sequel to the event. Such seizures are generally a result of oxygen toxicity, [25] [26] although hypoglycemia may be a contributing factor, but the latter risk can be eradicated or reduced by carefully monitoring the person's nutritional intake prior to oxygen treatment.

Oxygen first aid has been used as an emergency treatment for diving injuries for years. [27] Recompression in a hyperbaric chamber with the person breathing 100% oxygen is the standard hospital and military medical response to decompression illness. [27] [28] [29] The success of recompression therapy as well as a decrease in the number of recompression treatments required has been shown if first aid oxygen is given within four hours after surfacing. [30] There are suggestions that oxygen administration may not be the most effective measure for the treatment of decompression illness and that heliox may be a better alternative. [31]

Chronic obstructive pulmonary disease Edit

Care needs to be exercised in people with chronic obstructive pulmonary disease, such as emphysema, especially in those known to retain carbon dioxide (type II respiratory failure). Such people may further accumulate carbon dioxide and decreased pH (hypercapnation) if administered supplemental oxygen, possibly endangering their lives. [32] This is primarily as a result of ventilation–perfusion imbalance (see Effect of oxygen on chronic obstructive pulmonary disease). [33] In the worst case, administration of high levels of oxygen in people with severe emphysema and high blood carbon dioxide may reduce respiratory drive to the point of precipitating respiratory failure, with an observed increase in mortality compared with those receiving titrated oxygen treatment. [32] However, the risk of the loss of respiratory drive are far outweighed by the risks of withholding emergency oxygen, and therefore emergency administration of oxygen is never contraindicated. Transfer from field care to definitive care, where oxygen use can be carefully calibrated, typically occurs long before significant reductions to the respiratory drive.

A 2010 study has shown that titrated oxygen therapy (controlled administration of oxygen) is less of a danger to people with COPD and that other, non-COPD people, may also, in some cases, benefit more from titrated therapy. [32]

Fire risk Edit

Highly concentrated sources of oxygen promote rapid combustion. Oxygen itself is not flammable, but the addition of concentrated oxygen to a fire greatly increases its intensity, and can aid the combustion of materials (such as metals) which are relatively inert under normal conditions. Fire and explosion hazards exist when concentrated oxidants and fuels are brought into close proximity however, an ignition event, such as heat or a spark, is needed to trigger combustion. [34] A well-known example of an accidental fire accelerated by pure oxygen occurred in the Apollo 1 spacecraft in January 1967 during a ground test it killed all three astronauts. [35] A similar accident killed Soviet cosmonaut Valentin Bondarenko in 1961.

Combustion hazards also apply to compounds of oxygen with a high oxidative potential, such as peroxides, chlorates, nitrates, perchlorates, and dichromates because they can donate oxygen to a fire. [ relevant? ]

Concentrated O
2 will allow combustion to proceed rapidly and energetically. [34] Steel pipes and storage vessels used to store and transmit both gaseous and liquid oxygen will act as a fuel and therefore the design and manufacture of O
2 systems requires special training to ensure that ignition sources are minimized. [34] Highly concentrated oxygen in a high-pressure environment can spontaneously ignite hydrocarbons such as oil and grease, resulting in fire or explosion. The heat caused by rapid pressurization serves as the ignition source. For this reason, storage vessels, regulators, piping and any other equipment used with highly concentrated oxygen must be "oxygen-clean" prior to use, to ensure the absence of potential fuels. This does not apply only to pure oxygen any concentration significantly higher than atmospheric (approximately 21%) carries a potential risk.

Hospitals in some jurisdictions, such as the UK, now operate "no-smoking" policies, which although introduced for other reasons, support the aim of keeping ignition sources away from medical piped oxygen. Recorded sources of ignition of medically prescribed oxygen include candles, aromatherapy, medical equipment, cooking, and unfortunately, deliberate vandalism. Smoking of pipes, cigars, and cigarettes is of special concern. These policies do not entirely eliminate the risk of injury with portable oxygen systems, especially if compliance is poor. [36]

Alternative medicine Edit

Some practitioners of alternative medicine have promoted "oxygen therapy" as a cure for many human ailments including AIDS, Alzheimer's disease and cancer. The procedure may include injecting hydrogen peroxide, oxygenating blood, or administering oxygen under pressure to the rectum, vagina, or other bodily opening. [ citation needed ] According to the American Cancer Society, "available scientific evidence does not support claims that putting oxygen-releasing chemicals into a person's body is effective in treating cancer", and some of these treatments can be dangerous. [37]


Deletion of Psd1 and Pem2 dramatically alters mitochondrial PE/PC ratio

To dissect the roles of the two most abundant mitochondrial phospholipids, PE and PC, in MRC function and formation in vivo, we focused on psd1Δ and pem2Δ cells, which lack key enzymes for PE and PC biosynthesis, respectively (Figure 1). Previous studies showed that phospholipid composition and mitochondrial biogenesis is dependent upon the carbon source used in the growth medium (Tuller et al., 1999). Therefore we analyzed the phospholipid composition of yeast cells grown in glucose-containing, fermentable (SC glucose) or lactate-containing, nonfermentable (SC lactate) carbon sources. Whole-cell phospholipid analysis of glucose-grown psd1Δ cells revealed a threefold reduction in PE with a concomitant increase in PC (Figure 2A). Conversely, pem2Δ cells showed a 15-fold reduction in PC, with a twofold increase in PE and its precursor, phosphatidylmonomethylethanolamine (PMME), which could not be separated by the TLC used in this study (Figure 2A). Similar PE and PC changes were observed when psd1Δ and pem2Δ cells were grown in a medium containing lactate (Figure 2B). To analyze mitochondrial phospholipid composition, we obtained highly purified mitochondria with minimal contamination from other cellular organelles (Figure 2C). Consistent with whole-cell phospholipid composition, the levels of PE decreased by approximately sixfold in psd1Δ mitochondria (Figure 2D). The decrease in PE in psd1Δ mitochondria was accompanied by alterations in other phospholipids, including a significant increase in PC and PA and a decrease in CL (Figure 2D). In pem2Δ mitochondria, PC levels decreased fivefold, whereas the PE/PMME content doubled (Figure 2D). We also observed a significant accumulation of phosphatidyldimethylethanolamine in pem2Δ mitochondria (Figure 2D). In both glucose- and lactate-containing media, the absolute phospholipid levels in whole cells and in isolated mitochondria did not change in either mutant relative to the wild-type (WT) cells (Figure 2, E–G). These results suggest that in yeast cells, a homeostatic mechanism exists that buffers cells against the loss of the absolute amount of membrane phospholipids such that the depletion in PE is compensated by an increase in PC and vice versa. Therefore psd1Δ and pem2Δ cells have a significantly altered PE/PC ratio without any change in their absolute amount of membrane phospholipids. The mitochondrial PE/PC ratio in WT was 0.503, which was reduced to 0.065 in psd1Δ cells and increased to 6.02 in pem2Δ cells. Despite these dramatic deviations in the mitochondrial PE/PC ratios in psd1Δ and pem2Δ cells, the gross cellular and mitochondrial morphology was unaltered, with only a small reduction in the average length of mitochondrial cristae and outer membrane in psd1Δ cells (Supplemental Figure S1). There was no change in mitochondrial cristae length of pem2Δ cells, but we did observe a slight reduction in the average outer membrane length (Supplemental Figure S1). Collectively these results demonstrate that yeast cells can tolerate extensive alteration in mitochondrial PE/PC ratios and that a decrease in the PE level is countered by an increase in PC and vice versa.

FIGURE 2: Cellular and mitochondrial phospholipid composition of psd1Δ and pem2Δ cells. The whole-cell phospholipid composition of WT, psd1Δ, and pem2Δ cells grown in (A) SC glucose and (B) SC lactate. Phospholipid levels are expressed as the percentage of total phospholipid phosphorus in each phospholipid class. PE # represents the sum of PE and PMME in pem2Δ cells. Data are expressed as mean ± SD (n = 3) **p < 0.005, *p < 0.05. (C) Western blot analysis of crude and sucrose-gradient purified mitochondria from WT cells. Cox2, Dpm1, Pho8, and Pgk1 are used as markers of the yeast mitochondria, ER, vacuole, and cytoplasm, respectively. (D) Phospholipid composition of sucrose gradient–purified mitochondria from WT, psd1Δ, and pem2Δ cells grown in SC lactate. Data are expressed as mean ± SD (n = 3) **p < 0.005, *p < 0.05. (E, F) Total phospholipid content of whole-cell homogenates of WT, psd1Δ, and pem2Δ cells grown in (E) SC glucose or (F) SC lactate. (G) Total phospholipid content of mitochondria from SC lactate–grown cells. Data are expressed as mean ± SD (n = 3).

Decreased mitochondrial PE/PC ratio results in reduced respiration and ATP levels

To dissect the specific roles of PC and PE in MRC function, we performed extensive growth characterization of psd1Δ and pem2Δ cells in different carbon sources. The growth of psd1Δ and pem2Δ cells in fermentable SC glucose medium was comparable to that for WT cells (Figure 3A and Supplemental Figure S2A). Consistent with previous work (Birner et al., 2001), the growth of psd1Δ cells in nonfermentable SC lactate medium was severely compromised, whereas pem2Δ cells were able to grow, albeit at a slightly reduced rate (Figure 3B and Supplemental Figure S2B). The growth defects in nonfermentable medium suggested respiratory deficiency. To directly assess respiration, we measured oxygen consumption in WT, psd1Δ, and pem2Δ cells. Consistent with the severely diminished respiratory growth, the psd1Δ cells had a ∼60% reduction in oxygen consumption compared with WT cells (Figure 3, C and D). Oxygen consumption in pem2Δ cells was comparable to WT cells in SC lactate and even slightly elevated in SC glucose medium (Figure 3, C and D). The reduced growth of pem2Δ cells could be due to defects in mitochondrial protein import machinery, as reported recently (Schuler et al., 2015), and not to defects in respiration per se. In accordance with reduced respiration in psd1Δ cells, we observed a significant 50% reduction in ATP levels, whereas the ATP levels in the respiratory competent pem2Δ cells were unaltered (Figure 3, E and F). These results suggest that mitochondrial PE, but not PC, is essential for maintaining normal respiration.

FIGURE 3: Mitochondrial respiration is dependent on PE but not PC levels. Tenfold serial dilutions of WT, psd1Δ, and pem2Δ cells were spotted onto (A) SC glucose and (B) SC lactate plates, and images were captured after 2 (SC glucose) or 5 d (SC lactate) of growth at 30°C. Data are representative of at least three independent experiments. (C, D) WT, psd1Δ, and pem2Δ cells were grown in (C) SC glucose or (D) SC lactate to late log phase, and the rate of oxygen consumption was measured. Data are expressed as mean ± SD (n = 6) *p < 0.05, **p < 0.005. (E, F) Cellular ATP levels of WT, psd1Δ, and pem2Δ cells cultured in (E) SC glucose or (F) SC lactate. Data are expressed as mean ± SD (n = 3) *p < 0.05.

Decreased mitochondrial PE/PC ratio reduces MRC supercomplex activities without affecting supercomplex formation

To ascertain the biochemical basis for reduced respiration in PE-depleted cells, we analyzed the levels of native and denatured MRC complexes in the mitochondrial lysate from WT, psd1Δ, and pem2Δ cells grown in SC lactate medium. There was no change in the steady-state levels of individual MRC subunits (Supplemental Figure S3) or their incorporation into fully assembled MRC complexes in either of the mutant cells (Figure 4, A and B). As reported previously, MRC supercomplexes containing complexes III and IV were disrupted in CL-lacking crd1Δ cells (Zhang et al., 2002 Pfeiffer et al., 2003). These results imply that reduced PE and accompanying 33% decrease in CL levels in psd1Δ are insufficient to disrupt supercomplex formation. We noticed that pem2Δ cells, which exhibit an increased PE/PC ratio, showed an enhanced formation of a larger supercomplex (III2IV2) at the expense of the smaller supercomplex (III2IV Figure 4A). The lack of alterations in the amount and assembly of the MRC complexes cannot explain the respiratory deficiency of psd1Δ cells, suggesting that the reduced respiration could be due to a decrease in MRC activity. Therefore we measured the enzymatic activities of MRC complexes and observed four- and 2.5-fold reductions in complex III and IV activities, respectively, in psd1Δ cells (Figure 4, C and D). The specific reduction in complex III and IV activities could, in part, explain the respiratory defects observed in psd1Δ cells. The activities of MRC complexes were comparable in WT and pem2Δ cells (Figure 4, C and D), which is consistent with the normal respiratory phenotype of pem2Δ cells. Together these results demonstrate specific requirement of CL for MRC supercomplex formation and PE for MRC complex III and IV activities, whereas PC is redundant for these functions.

FIGURE 4: Mitochondrial PE is required for MRC complex III and IV activities but not MRC supercomplex formation. (A) Mitochondria from SC lactate–grown cells were solubilized by 1% digitonin and subjected to BN–PAGE/Western blot, and complexes II–V were detected by Sdh2, Rip1, Cox2, and Atp2 antibodies, respectively. Mitochondria from CL-deficient crd1Δ cells were used as positive control to demonstrate loss of supercomplexes (III2IV2, large supercomplex III2IV, small supercomplex) under identical conditions. (B) Samples from A were stained with Coomassie blue to demonstrate equal loading. (C) Digitonin-solubilized mitochondrial complexes from WT, psd1Δ, and pem2Δ cells were separated by CN-PAGE, followed by in-gel activity staining for complexes II–V. In-gel activities of MRC complexes were quantified by densitometric analysis, and relative activities were plotted for complexes II–V. Data were normalized to WT cells and expressed as mean ± SD (n = 3) **p < 0.005. (D) Samples from C were stained with Coomassie blue, and total protein, quantified using densitometric analysis, was used to normalize activity staining.

Depletion of PE results in a specific loss of mitochondrial DNA–encoded MRC subunits

In contrast to previous studies (Bottinger et al., 2012 Tasseva et al., 2013), which reported aberrant formation of MRC supercomplexes in PE depleted cells, we did not find any alterations in the MRC supercomplexes in psd1Δ cells. We reasoned that this discrepancy could be related to use of different growth conditions and carbon sources in these studies. Indeed, we found reduced levels of MRC supercomplexes (III2IV2 and III2IV) in glucose-grown psd1Δ cells (Figure 5A). These carbon source–dependent differences in MRC supercomplex assembly might be due to increased petite formation in psd1Δ cells, as reported previously (Birner et al., 2001). The petite phenotype results from mutations in the mitochondrial genome or loss of mitochondrial DNA (mtDNA), which leads to the loss of mtDNA-encoded MRC subunits. Consistent with the previous report, we found a significant increase in the number of petite colonies in the psd1Δ mutant (Supplemental Figure S4). Accordingly, SDS–PAGE/Western blot analysis of MRC subunits showed a specific decrease in the steady-state levels of mtDNA-encoded subunits Cox1, Cox2, and Cox3 (Figure 5B) without affecting the levels of the nuclear-encoded subunits Sdh2, Rip1, Cox4, and Atp2 (Figure 5C). Unlike PE-depleted psd1Δ cells, loss of PC in pem2Δ cells did not result in any alterations in the assembly or steady-state levels of MRC complexes or petite formation (Figure 5 and Supplemental Figure S4). Collectively these results suggest that the decrease in the MRC supercomplex levels in glucose-grown psd1Δ cells results from the loss of mtDNA-encoded subunits.

FIGURE 5: Depletion of mitochondrial PE in glucose-grown psd1Δ cells results in a specific loss of mtDNA-encoded MRC subunits. (A) Digitonin-solubilized mitochondria from SC glucose–grown WT, psd1Δ, and pem2Δ cells were subjected to BN–PAGE/Western blot. Complexes II–V were detected by Sdh2, Rip1, Cox2, and Atp2 antibodies, respectively. Data are representative of at least three independent experiments. (B) Mitochondria from SC glucose–grown WT, psd1Δ, and pem2Δ cells were subjected to SDS–PAGE, and mtDNA-encoded subunits were probed using Cox1, Cox2, and Cox3 antibodies. (C) Nuclear-encoded subunits were probed using Sdh2, Rip1, Cox4, and Atp2. Porin was used as a loading control. Data are representative of at least three independent experiments.

PE synthesized via Kennedy pathway completely rescues respiratory defects of psd1Δ cells by restoring mitochondrial PE levels

To determine whether PE synthesized in ER by the cytidine diphosphate–Etn branch of the Kennedy pathway could compensate for the loss of mitochondrial PE, we grew psd1Δ cells in the presence of Etn and measured cellular and mitochondrial phospholipids. Etn supplementation in psd1Δ cells completely restored cellular PE and significantly restored mitochondrial PE levels (Figure 6, A and B). Of interest, supplementation of Etn not only rescued mitochondrial PE levels, but it also restored PA and CL levels in psd1Δ mitochondria (Figure 6B), implying that a yet-unidentified homeostatic mechanism regulates the precise proportion of individual phospholipids in mitochondrial membranes. Next we asked whether the partial restoration of mitochondrial PE through exogenous Etn supplementation could restore the respiratory defects observed in psd1Δ cells. Indeed, Etn supplementation rescued the respiratory growth defect of psd1Δ cells in SC lactate medium (Figure 6, C and D). Consistent with the rescue of respiratory growth, Etn supplementation restored oxygen consumption (Figure 6E) and cellular ATP content (Figure 6F) in psd1Δ cells to WT levels. To investigate the mechanism by which Etn rescued respiration, we measured MRC complex III and IV activities and found that they were restored to WT levels in Etn-supplemented psd1Δ cells (Figure 6, G and H). Etn supplementation not only restored respiratory function in SC lactate, but it also rescued petite formation and cellular ATP levels in SC glucose medium (Supplemental Figure S5, A and B). Taken together, these results show that PE synthesized in ER by the Kennedy pathway can replenish mitochondrial PE and restore MRC complex III and IV activities in psd1Δ cells.

FIGURE 6: Ethanolamine supplementation rescues respiratory defects of psd1Δ cells by restoring mitochondrial PE levels. (A) Cellular and (B) mitochondrial phospholipid composition of WT cells grown in SC lactate and psd1Δ cells grown in SC lactate with and without 2 mM Etn. Phospholipid levels are expressed as percentage of total phospholipid phosphorus in each phospholipid class. Data are expressed as mean ± SD (n = 3) **p < 0.005, *p < 0.05. Tenfold serial dilutions of WT and psd1Δ cells were spotted onto (C) SC lactate and (D) SC lactate + 2 mM Etn plates, and images were captured after 4 d of growth at 30°C. Data are representative of at least three independent trials. (E) Rate of oxygen consumption and (F) total cellular ATP levels of WT and psd1Δ cells grown in SC lactate ± 2 mM Etn to late logarithmic phase were quantified. Data are expressed as mean ± SD (n = 3) *p < 0.05, **p < 0.005 (G, H) Digitonin- solubilized mitochondrial complexes from WT and psd1Δ cells grown in SC lactate ± 2 mM Etn were separated by CN-PAGE, followed by in-gel activity staining for (G) complex III and (H) complex IV. Densitometric quantifications of relative in-gel activities for complexes III and IV. Data were normalized to WT cells and are expressed as mean ± SD (n = 3) **p < 0.005, *p < 0.05. A.U., arbitrary units.

Endoplasmic reticulum–mitochondria encounter structure facilitates Etn-dependent rescue of mitochondrial PE deficiency

The complete rescue of mitochondrial bioenergetic phenotypes and respiratory growth of psd1Δ cells by Etn supplementation implies efficient transport of PE from ER to mitochondria (Figure 7A). To understand the molecular basis of PE import to mitochondria, we focused on the endoplasmic reticulum–mitochondria encounter structure (ERMES) complex, a mitochondria–ER tethering structure proposed to be involved in trafficking phospholipids between ER and mitochondria (Kornmann et al., 2009). First, to rule out the possibility that Etn itself or one of its metabolites is responsible for the psd1Δ rescue, we deleted the Kennedy pathway enzyme Ect1 in psd1Δ cells and showed that the rescue of psd1Δ cells by Etn is completely abrogated (Figure 7B). This result clearly demonstrates that PE synthesized via the Kennedy pathway is essential for psd1Δ rescue. Next we deleted two ERMES subunits, Mdm34 or Mdm12, both of which contain the synaptotagmin-like mitochondrial lipid-binding protein domain (SMP AhYoung et al., 2015), in psd1Δ cells and found that Etn rescue is reduced in the double mutants (Figure 7, C and D). To reveal the involvement of the ERMES complex in PE transport, we measured the phospholipid levels in mitochondria of psd1Δmdm34Δ cells with and without Etn supplementation. We did not observe any significant decrease in the steady levels of mitochondrial PE in the double mutant compared with psd1Δ single mutant after Etn supplementation (Supplemental Figure S6). These results suggest that ERMES is not essential for the import of nonmitochondrial PE to mitochondria but might only indirectly facilitate Etn-mediated rescue of psd1Δ cells.

FIGURE 7: PE synthesized by the Kennedy pathway requires ERMES for complete rescue of mitochondrial PE deficiency. (A) Schematic representation of the Kennedy pathway of PE biosynthesis and the ERMES complex. The Kennedy pathway enzyme Ect1, mitochondrial Psd1, and Mdm34 and Mdm12 of the ERMES complex are depicted in boldface to indicate that these genes are targeted to construct double-knockout strains. Tenfold serial dilutions of (B) WT, ect1Δ, psd1Δ, and psd1Δect1Δ, (C) WT, psd1Δ, mdm34Δ, and psd1Δmdm34Δ, and (D) WT, psd1Δ, mdm12Δ, and psd1Δmdm12Δ cells were spotted onto SC glucose and SC lactate ± Etn plates, and images were captured after 2 (SC glucose) or 5 d (SC lactate ± Etn) of growth at 30°C. Data are representative of at least three independent experiments.

6.1: Introduction to Oxygen Requirements - Biology

Daphnia is a frequently used food source in the freshwater larviculture (i.e. for different carp species) and in the ornamental fish industry (i.e. guppies, sword tails, black mollies and plattys etc.)

Daphnia belongs to the suborder Cladocera, which are small crustaceans that are almost exclusively living in freshwater. The carapace encloses the whole trunk, except the head and the apical spine (when present). The head projects ventrally and somewhat posteriorly in a beak-like snout. The trunk appendages (five or six pairs) are flattened, leaf-like structures that serve for suspension feeding (filter feeders) and for locomotion. The anterior part of the trunk, the postabdomen is turned ventrally and forward and bears special claws and spines to clean the carapace (Fig. 6.1.). Species of the genus Daphnia are found from the tropics to the arctic, in habitats varying in size from small ponds to large freshwater lakes. At present 50 species of Daphnia are reported worldwide, of which only six of them normally occur in tropical lowlands.

The adult size is subjected to large variations when food is abundant, growth continues throughout life and large adults may have a carapace length twice that of newly-mature individuals. Apart from differences in size, the relative size of the head may change progressively from a round to helmet-like shape between spring and midsummer. From midsummer to fall the head changes back to the normal round shape. These different forms are called cyclomorphs and may be induced, like in rotifers, by internal factors, or may be the result from an interaction between genetic and environmental conditions.

Normally there are 4 to 6 Instar stages Daphnia growing from nauplius to maturation through a series of 4-5 molts, with the period depending primarily on temperature (11 days at 10°C to 2 days at 25°C) and the availability of food. Daphnia species reproduce either by cyclical or obligate parthenogenesis and populations are almost exclusively female. Eggs are produced in clutches of two to several hundred, and one female may produce several clutches, linked with the molting process. Parthenogenetic eggs are produced ameiotically and result in females, but in some cases males can appear. In this way the reproductive pattern is similar to rotifers, where normally parthenogenetic diploid eggs are produced. The parthenogenetic eggs (their number can vary from 1 to 300 and depends largely upon the size of the female and the food intake) are laid in the brood chamber shortly after ecdysis and hatch just before the next ecdysis. Embryonic development in cladocerans occurs in the broodpouch and the larvae are miniature versions of the adults. In some cases the embryonic period does not correspond with the brood period, and this means that the larvae are held in the brood chamber even after the embryonic period is completed, due to postponed ecdysis (environmental factors). For different species the maturation period is remarkably uniform at given temperatures, ranging from 11 days at 10°C to only 2 days at 25°C.

Factors, such as change in water temperature or food depreviation as a result of population increase, may induce the production of males. These males have one or two gonopores, which open near the anus and may be modified into a copulatory organ. The male clasps the female with the first antennae and inserts the copulatory processes into the single, median female gonopore. The fertilized eggs are large, and only two are produced in a single clutch (one from each ovary), and are thick-shelled: these resting or dormant eggs being enclosed by several protective membranes, the ephippium. In this form, they are resistant to dessication, freezing and digestive enzymes, and as such play an important role in colonizing new habitats or in the re-establishment of an extinguished population after unfavourable seasonal conditions.

6.1.2. Nutritional value of Daphnia

The nutritional value of Daphnia depends strongly on the chemical composition of their food source. However, since Daphnia is a freshwater species, it is not a suitable prey organism for marine organisms, because of its low content of essential fatty acids, and in particular (n-3) HUFA. Furthermore, Daphnia contains a broad spectrum of digestive enzymes such, as proteinases, peptidases, amylases, lipases and even cellulase, that can serve as exo-enzymes in the gut of the fish larvae.

6.1.3. Feeding and nutrition of Daphnia

The filtering apparatus of Daphnia is constructed of specialized thoracic appendages for the collection of food particles. Five thoracic limbs are acting as a suction and pressure pump. The third and fourth pair of appendages carry large filter-like screens which filter the particles from the water. The efficiency of the filter allows even the uptake of bacteria (approx. 1µm). In a study on the food quality of freshwater phytoplankton for the production of cladocerans, it was found that from the spectrum blue-greens, flagellates and green algae, Daphnia performed best on a diet of the cryptomonads, Rhodomonas minuta and Cryptomonas sp., containing high levels of HUFA (more than 50% of the fatty acids in these two algae consisted of EPA and DHA, while the green algae were characterized by more 18:3n-3). This implies that the long-chained polyunsaturated fatty acids are important for a normal growth and reproduction of Daphnia . Heterotrophic microflagellates and ciliates up to the size of Paramecium can also be used as food for Daphnia . Even detritus and benthic food can be an important food source, especially when the food concentration falls below a certain threshold. In this case, the water current produced by the animals swimming on the bottom whirls up the material which is eventually ingested. Since daphnids seem to be non-selective filter feeders ( i.e., they do not discriminate between individual food particles by taste) high concentrations of suspended material can interfere with the uptake of food particles.

Figure 6.1. Schematic drawing of the internal and external anatomy of Daphnia.

6.1.4. Mass culture of Daphnia General procedure for tank culture

Daphnia is very sensitive to contaminants, including leaching components from holding facilities. When plastic or other polymer containers are used, a certain leaching period will be necessary to eliminate toxic compounds.

The optimal ionic composition of the culture medium for Daphnia is unknown, but the use of hard water, containing about 250 mg.l -1 of CO 3 2- , is recommended. Potassium and magnesium levels should be kept under 390 mg.l -1 and 30-240 µg. l -1 , respectively. Maintenance of pH between 7 to 8 appears to be important to successful Daphnia culture. To maintain the water hardness and high pH levels, lime is normally added to the tanks. The optimal culture temperature is about 25°C and the tank should be gently aerated to keep oxygen levels above 3.5 mg.l -1 (dissolved oxygen levels below 1.0 mg.l -1 are lethal to Daphnia ). Ammonia levels must be kept below 0.2 mg.l -1 .

Inoculation is carried out using adult Daphnia or resting eggs. The initial density is generally in the order of 20 to 100 animals per litre.

Normally, optimal algal densities for Daphnia culture are about 10 5 to 10 6 cells. ml -1 (larger species of Daphnia can support 10 7 to 10 9 -1 ). There are two techniques to obtain the required algal densities: the detrital system and the autotrophic system: Detrital system

The “stable tea” rearing system is a culture medium made up of a mixture of soil, manure and water. The manure acts as a fertilizer to promote algal blooms on which the daphnids feed. One can make use of fresh horse manure (200 g) that is mixed with sandy loam or garden soil (1 kg) in 10 l pond water to a stable stock solution this solution diluted two to four times can then be used as culture medium. Other fertilizers commonly used are: poultry manure (4 g.l -1 ) or cow-dung substrates. This system has the advantage to be self-maintaining and the Daphnia are not quickly subjected to deficiencies, due to the broad spectrum of blooming algae. However, the culture parameters in a detrital system are not reliable enough to culture Daphnia under standard conditions, i.e. overfertilization may occur, resulting in anoxic conditions and consequently in high mortalities and/or ephippial production. Autotrophic system

Autotrophic systems on the other hand use the addition of cultured algae. Green water cultures (10 5 to 10 6 -1 ) obtained from fish pond effluents are frequently used but these systems show much variation in production rate mainly because of the variable composition of algal species from one effluent to another. Best control over the culture medium is obtained when using pure algal cultures. These can be monocultures of e.g. algae such as Chlorella , Chlamydomonas or Scenedesmus , or mixtures of two algal cultures. The problem with these selected media is that they are not able to sustain many Daphnia generations without the addition of extra vitamins to the Daphnia cultures. A typical vitamin mix is represented in Table 6.1.

Table 6.1. A vitamin mix for the monospecific culture of Daphnia on Selenastrum, Ankistrodesmus or Chlamydomonas. One ml of this stock solution has to be added to each litre of algal culture medium (Goulden et al ., 1982).

Concentration of stock solution (µg.1 -1 )


To calculate the daily algal requirements and to estimate the harvesting time, regular sampling of the population density must be routinely undertaken. Harvesting techniques can be non-selective irrespective of size or age group, or selective (only the medium sized daphnids are harvested, leaving the neonates and matured individuals in the culture tank).

Mass cultivation of Daphnia magna can also be achieved on cheap agro-industrial residues, like cotton seed meal (17 g.l -1 ), wheat bran (6.7 g.l -1 ), etc . Rice bran has many advantages in comparison to other live foods (such as microalgae): it is always available in large quantities, it can be purchased easily at low prices, it can be used directly after simple treatment (micronisation, defatting), it can be stored for long periods, it is easy to dose, and it has none of the problems involved in maintenance of algal stocks and cultures.

In addition to these advantages, there is also the fact that rice bran has a high nutritional value rice bran (defatted) containing 24% (18.3%) crude protein, 22.8% (1.8%) crude fat, 9.2% (10.8%) crude fibre, and being a rich source of vitamins and minerals. Daphnia can be grown on this food item for an unlimited number of generations without noticeable deficiencies.

Defatted rice bran is preferred above raw rice bran because it prevents hydrolysis of the fatty acids present and, consequently, rancidity of the product. Micronisation of the bran into particles of less than 60 µm is generally carried out by treating an aqueous suspension (50 g.l -1 ) with a handmixer and filtering it through a 60 µm sieve, or by preparing it industrially by a dry mill process. The suspension is administered in small amounts throughout a 24 h period: 1 g of defatted rice bran per 500 individuals for two days (density: 100 animals.l -1 ). The food conversion ratio has an average of 1.7, which implies that with less than 2 kg of dry rice bran approximately 1 kg wet daphnid material can be produced (with a 25% water renewal per week De Pauw et al ., 1981). General procedure for pond culture

Daphnia can also be produced in ponds of at least 60 cm in height. To produce 1 ton of Daphnia biomass per week, a 2500 m 3 culture pond is required. The pond is filled with 5 cm of sun-dried (for 3 days) soil to which lime powder is added at a rate of 0.2 kg lime powder per ton soil. After this the pond is then filled with water up to 15 cm. Poultry manure is added to the ponds on the 4th day at a rate of 0.4 kg.m -3 to promote phytoplankton blooms. Fertilization of the pond with organic manure instead of mineral fertilizers is preferred because cladocerans can utilize much of the manure directly in the form of detritus. On day 12 the water level is raised to 50 cm and the pond is fertilized a second time with poultry manure (1 kg.m -3 ). Thereafter, weekly fertilization rates are maintained at 4 kg poultry manure per m -3 . In addition, fresh cow dung may also be used: in this instance a suspension is prepared containing 10 g.l -1 , which is then filtered through a 100 µm sieve. During the first week a 10 l extract is used per day per ton of water the fertilization increasing during the subsequent weeks from 20 l.m -3 .day -1 in the second week to 30 l.m -3 .day -1 in the following weeks.

The inoculation of the ponds is carried out on the 15th day at a rate of 10 daphnids per litre. One month after the inoculation, blooms of more than 100 g.m -3 can be expected. To maintain water quality in these ponds, fresh hard water can be added at a maximum rate of 25% per day. Harvesting is carried out by concentrating the daphnids onto a 500 µm sieve. The harvested biomass is concentrated in an aerated container (< 200 daphnids.l -1 ). In order to separate the daphnids from unfed substrates, exuviae and faecal material, the content of the container is brought onto a sieve, which is provided with a continuous circular water flow. The unfed particles, exuviae and faeces will collect in the centre on the bottom of the sieve, while the daphnids remain in the water column. The unwanted material can then be removed by using a pipette or sucking pump. Harvesting can be complete or partial for partial harvesting a maximum of 30% of the standing crop may be harvested daily. Contamination

Daphnia cultures are often accidentally contaminated with rotifers. In particular Brachionus, Conochilus and some bdelloids may be harmful, (i.e. B. rubens lives on daphnids and hinders swimming and food collection activities). Brachionus is simply removed from the culture by flushing the water and using a sieve of appropriate mesh size as Daphnia is much bigger than Brachionus . Conochilus , on the other hand, can be eliminated by adding cow dung to the culture (lowering the oxygen levels). Bdelloids are more difficult to remove from the culture since they are resistant to a wide range of environmental conditions and even drought. However, elimination is possible by creating strong water movements, which bring the bdelloids (which are bottom dwellers) in the water column, and then removing them by using sieves.

6.1.5. Production and use of resting eggs

Resting eggs are interesting material for storage, shipment and starting of new Daphnia cultures. The production of resting eggs can be initiated by exposing a part of the Daphnia culture to a combination of stressful conditions, such as low food availability, crowding of the animals, lower temperatures and short photoperiods. These conditions are generally obtained with aging populations at the end of the season. Collection of the ephippia from the wild can be carried out by taking sediment samples, rinsing them through a 200 µm sieve and isolating the ephippia under a binocular microscope. Normally, these embryos remain in dormancy and require a diapause inhibition to terminate this status, so that they can hatch when conditions are optimal. Possible diapause termination techniques are exposing the ephippia to low temperatures, darkness, oxygen and high carbon dioxide concentrations for a minimal period of several weeks (Davison, 1969).

There is still no standard hatching procedure for Daphnia. Generally the hatching process is stimulated by exposing the ephippia to higher temperatures (17-24°C), bright white light (70 W.m -2 ), longer photoperiods and high levels of dissolved oxygen. It is important, however, that these shocks are given while the resting eggs are still in the ephippium. After the shock the eggs may be removed from the ephippium. The hatching will then take place after 1-14 days.

6.1.6. Use of Moina

Moina also belongs to the Cladocera and many of the biological and cultural characteristics that have been discussed for Daphnia can be applied to Moina .

Moina thrives in ponds and reservoirs but primarily inhabits temporary ponds or ditches. The period to reach reproductive maturity takes four to five days at 26°C. At maturity clear sexual dimorphic characteristics can be observed in the size of the animals and the antennule morphology. Males (0.6-0.9 mm) are smaller than females (1.0-1.5 mm) and have long graspers which are used for holding the female during copulation. Sexually mature females carry only two eggs enclosed in an ephippium which is part of the dorsal exoskeleton.

Moina is of a smaller size than Daphnia , with a higher protein content, and of comparable economic value. Produced biomass is successfully used in the larviculture of rainbow trout, salmon, striped bass and by tropical fish hobbyists who also use it in a frozen form to feed over sixty fresh and salt water fish varieties. The partial replacement of Artemia by Moina micrura was also reported to have a positive effect during the larviculture of the freshwater prawn Macrobrachium rosenbergii (Alam, 1992).

Enrichment of Moina can be carried out using the direct method, by culturing them on baker’s yeast and emulsified fish or cuttlefish liver oils. Experiments have shown that Moina takes up (n-3) HUFA in the same way, although slower, than rotifers and Artemia nauplii, reaching a maximum concentration of around 40% after a 24 h-feeding period.

A Level Biology Project

This is an experiment to examine how the Surface Area / Volume Ratio affects the rate of diffusion in substrates and how this relates to the size and shape of living organisms.


This is an A-level biology project. It helped me get an A grade for biology many years ago. The whole project is reproduced here for your reference.

  • Aims
  • Background Information
  • Aparatus
  • Method
  • Prediction
  • Results
  • Interpretation
  • Precautions
  • Limitations
  • Anomolies
  • Extension Work

The surface area to volume ratio in living organisms is very important. Nutrients and oxygen need to diffuse through the cell membrane and into the cells. Most cells are no longer than 1mm in diameter because small cells enable nutrients and oxygen to diffuse into the cell quickly and allow waste to diffuse out of the cell quickly. If the cells were any bigger than this then it would take too long for the nutrients and oxygen to diffuse into the cell so the cell would probably not survive.

Single celled organisms can survive as they have a large enough surface area to allow all the oxygen and nutrients they need to diffuse through. Larger multi celled organisms need specialist organs to respire such as lungs or gills.

  1. Beakers
  2. Gelatin blocks containing cresol red dye
  3. 0.1M Hydrochloric acid
  4. Stop Watch
  5. Scalpel
  6. Tile
  7. Safety glasses

1. A block of gelatin which has been dyed with cresol red dye should be cut into blocks of the following sizes (mm).

5 x 5 x 5
10 x 10 x 10
15 x 15 x 15
20 x 20 x 20
10 x 10 x 2
10 x 10 x 10 (Triangle)
10 x 15 x 5
20 x 5 x 5

The triangle is of the following dimensions. [not reproduced]

The rest of the blocks are just plain cubes or rectangular blocks.

Cresol red dye is an acid / alkali indicator dye. In the alkali conditions of the gelatin it is red or purple but when it gets exposed to acid it turns a light yellow colour.

Gelatin is used for these tests as it is permeable and so it acts like a cell. It is easy to cut into the required sizes and the hydrochloric acid can diffuse at an even rate through it.

I am not using any blocks bigger than 20 x 20 x 20 as a preliminary test found that it was only practical to use blocks of 20mm³ or less as anything bigger than this would take longer than the amount of time that we have to do the experiment.

2. A small beaker was filled with 100cm³ of 0.1 molar Hydrochloric acid. This is a sufficient volume of acid to ensure that all the block sizes are fully covered in acid when dropped into the beaker.

3. One of the blocks is dropped into this beaker and the time for all the red dye to disappear is noted in a table such as the one below.

Dimensions (mm) Surface Area Volume (mm³) Surface Area / Volume Ratio Test 1 Test 2 Test 3 Average Time

4. This test should be repeated for all the sizes of blocks three times to ensure a fair test. Fresh acid should be used for each block to ensure that this does not affect the experiment’s results.

5. The surface area / volume ratio and an average of the results can then be worked out. A graph of Time against Surface Area to Volume Ratio can then be plotted. From this graph we will be able to see how the surface area affects the time taken for the hydrochloric acid to penetrate to the centre of the cube.

I predict that as the Surface Area / Volume Ratio increases the time taken for the hydrochloric acid to penetrate to the centre of the cube will go down. This is because a small block has a large amount of surface area compared to it’s volume so the hydrochloric acid will have a large surface area to diffuse through. A larger block has a smaller amount of surface area in relation to it’s size so it should take longer for the hydrochloric acid to diffuse into the centre of the cube. The actual rate of the hydrochloric acid diffusing through the gelatin should be the same for all the blocks but when the surface area / volume ratio goes up it will take less time for the hydrochloric acid to reach the centre of the cube.

I carried out the above experiment and these results were obtained.

Dimensions (mm) Surface Area Volume (mm³) Surface Area / Volume Ratio Test 1 Test 2 Test 3 Average Time
5 x 5 x 5 150 125 1.2:1 7.02 6.57 4.53 6.16
10 x 10 x 10 600 1,000 0.6:1 10.3 23.25 15.33 16.28
15 x 15 x 15 1,350 3,375 0.4:1 29.55 30.22 23.45 28.01
20 x 20 x 20 2,400 8,000 0.3:1 53.4 32.44 58.56 48.3
10 x 10 x 2 280 200 1.4:1 0.26 0.37 1.58 1.01
10 x 15 x 5 550 750 0.73:1 7.2 10.23 10.47 9.3
20 x 5 x 5 450 500 0.9:1 3.18 2.58 4.09 3.29
10 x 10 x 10
441.42 500 0.88:1 9.58 3.34 5.25 6.19

The Surface area to Volume ratio is calculated by

Surface Area To Volume Ratio = Surface Area / Volume

From these rates I was able to plot a graph of the Surface Area to Volume Ratio against time.

In all the blocks of gelatin the rate of penetration of the hydrochloric acid from each side would have been the same but all the blocks take different amounts of time to clear because they are different sizes. As the blocks get bigger it takes longer for the hydrochloric acid to diffuse through all the block and so clear the dye. It takes longer to reach the centre of the cube even though the rate of diffusion is the same for all the cubes.

As the volume of the blocks goes up the Surface Area / Volume ratio goes down. The larger blocks have a smaller proportion of surface area than the smaller blocks. The smallest block has 1.4mm² of surface area for every 1mm³ of volume. The largest block only has 0.3mm² of surface area for each 1mm³ of volume. This means that the hydrochloric acid is able to diffuse to the centre of the smallest block much faster than the largest block. The acid took 48 minutes to diffuse to the centre of the largest block but only 1 minute in the smallest block. A living cell would not survive if it had to wait 48 minutes for oxygen to diffuse through it so living cells need to be very small.

When the surface area to volume ratio goes down it takes longer for the hydrochloric acid to diffuse into the cube but if the ratio goes up then the hydrochloric acid diffuses more quickly into the block of gelatin. Some shapes have a larger surface area to volume ratio so the shape of the object can have an effect on the rate of diffusion.

It is important that cells have a large surface area to volume ratio so that they can get enough nutrients into the cell. They can increase their surface area by flattening and becoming longer or by having a rough surface with lots of folds of cell membrane known as villi. [picture not reproduced]

The villi vastly increase the surface area of the cell whereas the cell which is round only has a small surface area in relation to it’s volume. Both cells above have an volume of 1cm³. The cell on the left has a surface area of 3cm² but the cell on the right with villi has a surface area of 10cm². The cell membrane is made up of a lipid bi-layer with many proteins integrated into it. [picture not reproduced]

Oxygen can diffuse easily through the membrane and Carbon Dioxide and other waste products can easily dissolve out. The concentration of oxygen in the cell is always lower than outside the cell which causes the oxygen to diffuse in. Gases will always dissolve from an area of high to low pressure. The concentration of carbon dioxide outside the cell is lower than the concentration in the cell so the carbon dioxide will always dissolve out of the cell.

Single celled organisms such as amoebas have a large surface area to volume ratio because they are so small. They are able to get all the oxygen and nutrients they need by diffusion through the cell membrane.

Larger organisms such as mammals have a relatively small surface area compared to their volume so they need special systems such as the lungs in order to get enough oxygen. Surface area to volume ratio is very important in lungs where a large amount of oxygen has to get into the lungs. The lungs have a very large surface area because they contain millions of sacs called alveoli which allow oxygen to diffuse into the bloodstream. By having millions of these alveoli the lungs are able to cram a very large surface area into a small space. This surface area is sufficient for all the oxygen we need to diffuse through it and to let the carbon dioxide out.

By increasing the surface area the rate of diffusion will go up.

a) All the gelatin used should be taken from the same block to ensure that all the blocks are made up of the same materials.

b) All the tests should be done at room temperature to ensure that the blocks of gelatin do not melt.

c) The same volume of acid should be used for all the tests to ensure that the rate of diffusion can not be affected by the pressure of a larger volume of acid.

d) Safety glasses should be worn to protect your eyes from the hydrochloric acid.

To help make this experiment more accurate, I repeated it three times for each block size and then used the average of all the results to plot a graph with a line of best fit. I tried to keep all the variables except for the size of the gelatin blocks the same for all the experiments. However, in reality it is impossible to keep all the variables precisely the same. For example:

a) It is also impossible to precisely measure the size of gelatin block each time. I measured the sizes to the nearest mm so the sizes of block that I used should be correct to the nearest mm.

b) When the gelatin blocks are dropped into the beakers the base of the block comes into contact with the bottom of the beaker which reduces the surface area of the block that comes into contact with the hydrochloric acid.

c) The results will be slightly inaccurate as the moment when the gelatin block has lost all it’s dye is a matter of opinion and not something that can be measured precisely.

d) Due to the fairly slow speed of our reactions it is only possible to measure the time of the reaction to the nearest 0.1 second even though the stopwatch shows the measurements to the nearest 0.01 second.

The graph produced shows a smooth curve with a decreasing gradient as the surface area to volume ratio goes up. The only anomaly is the result for the 5 x 5 x 5 block. The result here is higher than the curve of best fit for the graph. The results for the 5 x 5 x 5 block ranged from 4.53 to 7.02 seconds with an average of 6.15 seconds. The line of best fit for the graph suggests that the average should be around 3 seconds. The anomalous result was probably due to experimental error as a result of this being the first block size that I used in the experiment. The most likely explanation is that I was unsure of how to judge when all the dye had disappeared and as a result delayed pressing the stop button of the stop watch. As the experiment progressed with the other block sizes I probably got better at making this judgement.

This experiment could be improved in a number of ways.

1) It could be repeated more times to help get rid of any anomalies. A better overall result would be obtained by repeating the experiment more times because any errors in one experiment should be compensated for by the other experiments.

2) Using more shapes and sizes of gelatin block would have produced a better looking graph.

3) Variables that might affect the rate of diffusion could be investigated. The rate of diffusion may also be affected by temperature, strength of acid and volume of acid

4) The block could be suspended in the hydrochloric acid so than none of it’s surfaces are in contact with the wall of the beaker. A small cradle could be used to suspend the blocks in the acid which would mean that all six sides of the cube should be in contact with the acid. This would ensure that diffusion could occur evenly through all the sides of the cube.


This is a real A-level school project and as such is intended for educational or research purposes only. Extracts of this project must not be included in any projects that you submit for marking. Doing this could lead to being disqualified from all the subjects that you are taking. You have been warned. If you want more help with doing your biology practicals then have a look at 'Advanced Level Practical Work for Biology' by Sally Morgan. If you want more detailed biology information then I'd recommend the book 'Advanced Biology' by M. Kent.


This entry was posted on Monday, June 2nd, 2008 at 8:14 pm and is filed under Life. You can follow any responses to this entry through the RSS 2.0 feed. You can leave a response, or trackback from your own site.

2 Responses to “Surface Area / Volume Ratio Biology Experiment”

How do u make the derail red & gelatin cubes? I have failed at using powdered gelatin & so was wondering what to use & what ratio?

I would like to quote this page in a similar experiment i am doing in my biology class, is that ok?


Several studies indicate that aerobes can survive in the presence of oxygen only by virtue of an elaborate system of defenses. Without these defenses, key enzyme systems in the organisms fail to function and the organisms die.
Obligate anaerobes, which live only in the absence of oxygen, do not possess the defenses that make aerobic life possible and therefore can not survive in air.

The tolerance to oxygen is related to the ability of the bacterium to detoxify superoxide and hydrogen peroxide, produced as a byproduct of aerobic respiration.

The assimilation of glucose in aerobic conditions results in the terminal generation of free radical superoxide (O2 – ). The superoxide is reduced by the enzyme superoxide dismutase to oxygen gas and hydrogen peroxide (H2O2). Subsequently, the toxic hydrogen peroxide generated in this reaction is converted to water and oxygen by the enzyme catalase, which is found in aerobic and facultative bacteria, or by various peroxidases which are found in several aerotolerant anaerobes.

Obligate aerobes and most facultative anaerobes have both superoxide dismutase and catalase. Some facultative and aerotolerant anaerobes have superoxide dismutase but lack catalase. Most obligate anaerobes lack both enzymes.

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