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Investigating an enzyme-controlled reaction: catalase and hydrogen peroxide concentration, class practical or demonstration.

Hydrogen peroxide ( H 2 O 2 ) is a by-product of respiration and is made in all living cells. Hydrogen peroxide is harmful and must be removed as soon as it is produced in the cell. Cells make the enzyme catalase to remove hydrogen peroxide.

This investigation looks at the rate of oxygen production by the catalase in pureed potato as the concentration of hydrogen peroxide varies. The oxygen produced in 30 seconds is collected over water. Then the rate of reaction is calculated.

Lesson organisation

You could run this investigation as a demonstration at two different concentrations, or with groups of students each working with a different concentration of hydrogen peroxide. Individual students may then have time to gather repeat data. Groups of three could work to collect results for 5 different concentrations and rotate the roles of apparatus manipulator, result reader and scribe. Collating and comparing class results allows students to look for anomalous and inconsistent data.

Apparatus and Chemicals

For each group of students:.

Pneumatic trough/ plastic bowl/ access to suitable sink of water

Conical flask, 100 cm 3 , 2

Syringe (2 cm 3 ) to fit the second hole of the rubber bung, 1

Measuring cylinder, 100 cm 3 , 1

Measuring cylinder, 50 cm 3 , 1

Clamp stand, boss and clamp, 2

Stopclock/ stopwatch

For the class – set up by technician/ teacher:

Hydrogen peroxide, range of concentrations, 10 vol, 15 vol, 20 vol, 25 vol, and 30 vol, 2 cm 3 per group of each concentration ( Note 1 )

Pureed potato, fresh, in beaker with syringe to measure at least 20 cm 3 , 20 cm 3 per group per concentration of peroxide investigated ( Note 2 )

Rubber bung, 2-holed, to fit 100 cm 3 conical flasks – delivery tube in one hole (connected to 50 cm rubber tubing)

Health & Safety and Technical notes

Wear eye protection and cover clothing when handling hydrogen peroxide. Wash splashes of pureed potato or peroxide off the skin immediately. Be aware of pressure building up if reaction vessels become blocked. Take care inserting the bung in the conical flask – it needs to be a tight fit, so push and twist the bung in with care.

Read our standard health & safety guidance

1 Hydrogen peroxide: (See CLEAPSS Hazcard) Solutions less than 18 vol are LOW HAZARD. Solutions at concentrations of 18-28 vol are IRRITANT. Take care when removing the cap of the reagent bottle, as gas pressure may have built up inside. Dilute immediately before use and put in a clean brown bottle, because dilution also dilutes the decomposition inhibitor. Keep in brown bottles because hydrogen peroxide degrades faster in the light. Discard all unused solution. Do not return solution to stock bottles, because contaminants may cause decomposition and the stock bottle may explode after a time.

2 Pureed potato may irritate some people’s skin. Make fresh for each lesson, because catalase activity reduces noticeably over 2/3 hours. You might need to add water to make it less viscous and easier to use. Discs of potato react too slowly.

3 If the bubbles from the rubber tubing are too big, insert a glass pipette or glass tubing into the end of the rubber tube.

SAFETY: Wear eye protection and protect clothing from hydrogen peroxide. Rinse splashes of peroxide and pureed potato off the skin as quickly as possible.

Preparation

a Make just enough diluted hydrogen peroxide just before the lesson. Set out in brown bottles ( Note 1 ).

b Make pureed potato fresh for each lesson ( Note 2 ).

c Make up 2-holed bungs as described in apparatus list and in diagram.

Investigation

d Use the large syringe to measure 20 cm 3 pureed potato into the conical flask.

e Put the bung securely in the flask – twist and push carefully.

f Half-fill the trough, bowl or sink with water.

g Fill the 50 cm 3 measuring cylinder with water. Invert it over the trough of water, with the open end under the surface of the water in the bowl, and with the end of the rubber tubing in the measuring cylinder. Clamp in place.

h Measure 2 cm 3 of hydrogen peroxide into the 2 cm 3 syringe. Put the syringe in place in the bung of the flask, but do not push the plunger straight away.

i Check the rubber tube is safely in the measuring cylinder. Push the plunger on the syringe and immediately start the stopclock.

j After 30 seconds, note the volume of oxygen in the measuring cylinder in a suitable table of results. ( Note 3 .)

k Empty and rinse the conical flask. Measure another 20 cm 3 pureed potato into it. Reassemble the apparatus, refill the measuring cylinder, and repeat from g to j with another concentration of hydrogen peroxide. Use a 100 cm 3 measuring cylinder for concentrations of hydrogen peroxide over 20 vol.

l Calculate the rate of oxygen production in cm 3 /s.

m Plot a graph of rate of oxygen production against concentration of hydrogen peroxide.

Teaching notes

Note the units for measuring the concentration of hydrogen peroxide – these are not SI units. 10 vol hydrogen peroxide will produce 10 cm 3 of oxygen from every cm 3 that decomposes.( Note 1 .)

In this procedure, 2 cm 3 of 10 vol hydrogen peroxide will release 20 cm 3 of oxygen if the reaction goes to completion. 2 cm 3 of liquid are added to the flask each time. So if the apparatus is free of leaks, 22 cm 3 of water should be displaced in the measuring cylinder with 10 vol hydrogen peroxide. Oxygen is soluble in water, but dissolves only slowly in water at normal room temperatures.

Use this information as a check on the practical set-up. Values below 22 cm 3 show that oxygen has escaped, or the hydrogen peroxide has not fully reacted, or the hydrogen peroxide concentration is not as expected. Ask students to explain how values over 22 cm 3 could happen.

An error of ± 0.05 cm 3 in measuring out 30 vol hydrogen peroxide could make an error of ± 1.5 cm 3 in oxygen production.

Liver also contains catalase, but handling offal is more controversial with students and introduces a greater hygiene risk. Also, the reaction is so vigorous that bubbles of mixture can carry pieces of liver into the delivery tube.

If collecting the gas over water is complicated, and you have access to a 100 cm 3 gas syringe, you could collect the gas in that instead. Be sure to clamp the gas syringe securely but carefully.

The reaction is exothermic. Students may notice the heat if they put their hands on the conical flask. How will this affect the results?

Health and safety checked, September 2008

http://www.saps.org.uk/secondary/teaching-resources/293-student-sheet-24-microscale-investigations-with-catalase Microscale investigations with catalase – which has been transcribed onto this site at Investigating catalase activity in different plant tissues.

(Website accessed October 2011)

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Science project, catalase and hydrogen peroxide experiment.

How do living cells interact with the environment around them? All living things possess catalysts , or substances within them that speed up chemical reactions and processes. Enzymes are molecules that enable the chemical reactions that occur in all living things on earth. In this catalase and hydrogen peroxide experiment, we will discover how enzymes act as catalysts by causing chemical reactions to occur more quickly within living things. Using a potato and hydrogen peroxide, we can observe how enzymes like catalase work to perform decomposition , or the breaking down, of other substances. Catalase works to speed up the decomposition of hydrogen peroxide into oxygen and water. We will also test how this process is affected by changes in the temperature of the potato. Is the process faster or slower when compared to the control experiment conducted at room temperature?

What happens when a potato is combined with hydrogen peroxide?

• Hydrogen peroxide
• Small glass beaker or cup
• Divide the potato into three roughly equal sections.
• Keep one section raw and at room temperature.
• Place another section in the freezer for at least 30 minutes.
• Boil the last section for at least 5 minutes.
• Chop and mash a small sample (about a tablespoon) of the room temperature potato and place into beaker or cup.
• Pour enough hydrogen peroxide into the cup so that potato is submerged and observe.
• Repeat steps 5 & 6 with the boiled and frozen potato sections.

Observations & Results

Watch each of the potato/hydrogen peroxide mixtures and record what happens. The bubbling reaction you see is the metabolic process of decomposition , described earlier. This reaction is caused by catalase, an enzyme within the potato. You are observing catalase breaking hydrogen peroxide into oxygen and water. Which potato sample decomposed the most hydrogen peroxide? Which one reacted the least?

You should have noticed that the boiled potato produced little to no bubbles. This is because the heat degraded the catalase enzyme, making it incapable of processing the hydrogen peroxide. The frozen potato should have produced fewer bubbles than the room temperature sample because the cold temperature slowed the catalase enzyme’s ability to decompose the hydrogen peroxide. The room temperature potato produced the most bubbles because catalase works best at a room temperature.

Conclusions

Catalase acts as the catalyzing enzyme in the decomposition of hydrogen peroxide. Nearly all living things possess catalase, including us! This enzyme, like many others, aids in the decomposition of one substance into another. Catalase decomposes, or breaks down, hydrogen peroxide into water and oxygen.

Want to take a closer look? Go further in this experiment by looking at a very small sample of potato combined with hydrogen peroxide under a microscope!

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Testing for catalase enzymes

In association with Nuffield Foundation

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Try this class experiment to detect the presence of enzymes as they catalyse the decomposition of hydrogen peroxide

Enzymes are biological catalysts which increase the speed of a chemical reaction. They are large protein molecules and are very specific to certain reactions. Hydrogen peroxide decomposes slowly in light to produce oxygen and water. The enzyme catalase can speed up (catalyse) this reaction.

In this practical, students investigate the presence of enzymes in liver, potato and celery by detecting the oxygen gas produced when hydrogen peroxide decomposes. The experiment should take no more than 20–30 minutes.

• Eye protection
• Conical flasks, 100 cm 3 , x3
• Measuring cylinder, 25 cm 3
• Bunsen burner
• Wooden splint
• A bucket or bin for disposal of waste materials
• Hydrogen peroxide solution, ‘5 volume’

Health, safety and technical notes

• Read our standard health and safety guidance.
• Wear eye protection throughout. Students must be instructed NOT to taste or eat any of the foods used in the experiment.
• Hydrogen peroxide solution, H 2 O 2 (aq) – see CLEAPSS Hazcard HC050  and CLEAPSS Recipe Book RB045. Hydrogen peroxide solution of ‘5 volume’ concentration is low hazard, but it will probably need to be prepared by dilution of a more concentrated solution which may be hazardous.
• Only small samples of liver, potato and celery are required. These should be prepared for the lesson ready to be used by students. A disposal bin or bucket for used samples should be provided to avoid these being put down the sink.
• Measure 25 cm 3  of hydrogen peroxide solution into each of three conical flasks.
• At the same time, add a small piece of liver to the first flask, a small piece of potato to the second flask, and a small piece of celery to the third flask.
• Hold a glowing splint in the neck of each flask.
• Note the time taken before each glowing splint is relit by the evolved oxygen.
• Dispose of all mixtures into the bucket or bin provided.

Teaching notes

Some vegetarian students may wish to opt out of handling liver samples, and this should be respected.

Before or after the experiment, the term enzyme will need to be introduced. The term may have been met previously in biological topics, but the notion that they act as catalysts and increase the rate of reactions may be new. Similarly their nature as large protein molecules whose catalytic activity can be very specific to certain chemical reactions may be unfamiliar. The name catalase for the enzyme present in all these foodstuffs can be introduced.

To show the similarity between enzymes and chemical catalysts, the teacher may wish to demonstrate (or ask the class to perform as part of the class experiment) the catalytic decomposition of hydrogen peroxide solution by manganese(IV) oxide (HARMFUL – see CLEAPSS Hazcard HC060).

If students have not performed the glowing splint test for oxygen for some time, they may need reminding of how to do so by a quick demonstration by the teacher.

More resources

Add context and inspire your learners with our short career videos showing how chemistry is making a difference .

Additional information

This is a resource from the  Practical Chemistry project , developed by the Nuffield Foundation and the Royal Society of Chemistry.

Practical Chemistry activities accompany  Practical Physics  and  Practical Biology .

© Nuffield Foundation and the Royal Society of Chemistry

• 11-14 years
• 14-16 years
• 16-18 years
• Practical experiments
• Biological chemistry

Specification

• Enzymes act as catalysts in biological systems.
• Factors which affect the rates of chemical reactions include: the concentrations of reactants in solution, the pressure of reacting gases, the surface area of solid reactants, the temperature and the presence of catalysts.
• Describe the characteristics of catalysts and their effect on rates of reaction.
• Recall that enzymes act as catalysts in biological systems.
• 7.6 Describe a catalyst as a substance that speeds up the rate of a reaction without altering the products of the reaction, being itself unchanged chemically and in mass at the end of the reaction
• 7.8 Recall that enzymes are biological catalysts and that enzymes are used in the production of alcoholic drinks
• C6.2.4 describe the characteristics of catalysts and their effect on rates of reaction
• C6.2.5 identify catalysts in reactions
• C6.2.14 describe the use of enzymes as catalysts in biological systems and some industrial processes
• C5.2f describe the characteristics of catalysts and their effect on rates of reaction
• C5.2i recall that enzymes act as catalysts in biological systems
• C6.2.13 describe the use of enzymes as catalysts in biological systems and some industrial processes
• C5.1f describe the characteristics of catalysts and their effect on rates of reaction
• C5.1i recall that enzymes act as catalysts in biological systems
• B2.24 The action of a catalyst, in terms of providing an alternative pathway with a lower activation energy.
• 2.3.5 demonstrate knowledge and understanding that a catalyst is a substance which increases the rate of a reaction without being used up and recall that transition metals and their compounds are often used as catalysts;
• 7. Investigate the effect of a number of variables on the rate of chemical reactions including the production of common gases and biochemical reactions.

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Hydrogen Peroxide

Safety and handling .

Safety has always been one of Evonik's main concerns, and not only when handling hydrogen peroxide safe. As we are clearly committed to the chemical industry's Responsible Care program, we strive to achieve the highest possible level of safety and quality for hydrogen peroxide both in our own facilities and laboratories and for our customers.

On this page we have summarized the safety risks associated with hydrogen peroxide (H 2 O 2 ), its handling and storage . Today, many risks are rather unlikely, as there are globally accepted technical standards. 

Nevertheless, our customers should be aware of the risks and understand the need for certain precautions when working with hydrogen peroxide.

Hydrogen peroxide safety and potential risks

Hydrogen peroxide is a clear colorless liquid, which in appearance resembles water. Therefore, spilled product or hydrogen peroxide in unlabelled containers could erroneously be regarded as water. It is highly corrosive to the skin and eyes. It is a strong oxidizing chemical and, therefore, tends to react rapidly, sometimes even violently with various substances, such as several metals, leather or alkali reagents.

Hydrogen peroxide solutions themselves are not flammable. Highly concentrated hydrogen peroxide, however, can ignite inflammable materials, and the oxygen released by decomposition additionally promotes the combustion. Even at low concentrations, ignition can occur under unfavorable conditions after a gradual concentration of the hydrogen peroxide due to the preferred evaporation of water. 

Vapors can explode if the hydrogen peroxide concentration in the vapor phase is higher than 26 mol% (40%w/w). Explosions are ignited by sparks, contact with a catalytically active material, or – at temperatures above 150 °C – even by catalytically non-active materials. At normal pressure, such vapor compositions can only occur if the hydrogen peroxide concentration of the liquid is 74 wt% or higher and the temperature of the liquid is higher than 100 °C. 

Explosive and shock-sensitive mixtures can be formed if concentrated hydrogen peroxide comes into contact with organic compounds. According to literature data, there is a general risk of detonations if the content of hydrogen peroxide in the resulting mixture is 25% by weight or above. In any case, appropriate safety precautions must be taken to avoid critical conditions.

H 2 O 2 first aid brochure

Any person with hydrogen peroxide should be familiar with personal protection measures, emergency procedures and first aid. Exposed persons should act or be treated according to the recommendations in this brochure, and, if in doubt, consult a doctor. ... MORE

Training for safe handling required

As a consequence of the hazards resulting from the properties of hydrogen peroxide, it is imperative that all personnel handling hydrogen peroxide receive proper training and instructions for safe handling, first aid and emergency response, which meet the local regulatory standards as well as special requirements. The most essential rules for the handling of hydrogen peroxide are summarized in the table below.

Guidelines and rules for a safe handling of hydrogen peroxide

For further information please see the MSDS/SDS of your specific product, available from your local Evonik representative . If you require any additional advice please also don’t hesitate to get in touch with your local Evonik expert.

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Safety Video

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Hydrogen peroxide training video 

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Top 7 Science Experiments with Hydrogen Peroxide

Welcome to our carefully curated collection of hydrogen peroxide science experiments. This roundup invites you to journey through experiments showing you this simple compound’s versatile nature.

Hydrogen peroxide is a common household item known for its antiseptic properties. Yet, beneath its seemingly mundane identity lies a treasure trove of chemical wonders waiting to be explored. With its reactive nature and ability to break down into simpler molecules, hydrogen peroxide is a captivating subject for many scientific experiments.

Note : Students should know the concentration of hydrogen peroxide and understand its potential hazards. These experiments should be conducted in a controlled manner, adhering to the provided procedure and under the supervision of an adult.

1. Elephant Toothpaste

One experiment that is sure to captivate the minds of both students and teachers alike is the famous “Elephant Toothpaste” experiment using hydrogen peroxide.

Elephant Toothpaste experiment is a must-try for any classroom, sparking excitement and curiosity while reinforcing fundamental chemistry principles.

2. Genie in a Bottle

This experiment is an absolute must-try for students, as it offers a hands-on journey into the world of chemical reactions.

By delving into “Genie in a Bottle,” you’ll unleash your curiosity, hone critical thinking skills, and witness the power of chemistry firsthand.

3. DIY Pasta Rocket Engine

The DIY Pasta Rocket Engine experiment using hydrogen peroxide (H2O2) is a captivating and exciting activity that students and teachers should definitely try.

This experiment provides an excellent opportunity for students to explore the principles of chemical reactions, combustion, and propulsion in a hands-on and engaging manner.

4. Remove Stains Using Hydrogen Peroxide

Learning how to remove stains using hydrogen peroxide is a practical and useful experiment that both students and teachers should try. Hydrogen peroxide possesses excellent stain-removing properties due to its oxidizing nature, making it a valuable tool for tackling a wide range of stains.

5. Flame Light Relight – Science Magic

The Flame Light Relight experiment is an intriguing and educational experience that students and teachers should approach with caution.

By engaging in the Flame Light Relight experiment responsibly, students can gain a deeper understanding of the science behind fire and chemical reactions while reinforcing the importance of safety measures and responsible experimentation.

Learn more: Flame Light Relight

6. Potato Catalyzed H2O2 Decomposition

The Potato Catalyzed H2O2 Decomposition experiment is a fascinating and educational activity that students and teachers should definitely try. In this experiment, the natural enzymes present in a potato act as a catalyst to accelerate the decomposition of hydrogen peroxide.

7. Boiled Versus Fresh Liver with Hydrogen Peroxide

The Boiled Versus Fresh Liver with Hydrogen Peroxide experiment is a captivating and informative activity that students and teachers should consider trying.

By comparing the reaction of hydrogen peroxide with boiled and fresh liver, students can explore the effects of heat on enzymatic activity.

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Hydrogen Peroxide Health & Safety Tips

06/17/2015 Posted on June 17, 2015 | in Safety

What is Hydrogen Peroxide?

Hydrogen peroxide was first discovered in the early 19 th century, but the pure chemical was not produced until 1894. Today, this chemical is produced in quantities of more than 2 million tons worldwide each year.

Hydrogen peroxide is a chemical compound usually produced as a diluted solution . This colorless, slightly viscous chemical has a somewhat sharp odor. Pure hydrogen peroxide poses some extreme health risks, and diluted solutions of 3-30 percent can cause potential hazards as well. As a result, hydrogen peroxide should be handled and stored with care whether at home or in the workplace.

Read on for safety tips about handling and storing this chemical safely, and how to manage accidental exposure.

Common Uses of Hydrogen Peroxide

Hydrogen peroxide is available most commonly as an aqueous solution, usually at 3 or 6 percent concentrations for household use. In laboratories, 30 percent hydrogen peroxide is used. Commercial-grade peroxide at as high as 98 percent purity is also available, but it’s important to remember that the higher the concentration, the higher the potential health risks. This chemical is used in a variety of applications, including:

• Bleaching paper and pulp
• Mild laundry detergent bleach
• Disinfectant
• Cosmetic hair bleach
• Flour bleaching agent
• Acne treatment
• Wastewater treatment

Hazards Associated with Hydrogen Peroxide

In high concentrations in laboratory or industrial settings, hydrogen peroxide can pose serious health and safety hazards. Hydrogen peroxide is a strong oxidizer (moderate oxidizer in lower concentrations), and can be  corrosive to the eyes, skin, and respiratory system. This chemical can cause burns to the skin and tissue damage to the eyes.

Take special caution to avoid contact with hydrogen peroxide mist. Household-grade concentrations of this chemical are generally considered safe to use, but should never be ingested. Due to these potential hazards, hydrogen peroxide should be handled with care.

Hydrogen Peroxide Safety, Handling & First Aid

When handling moderate-to-high concentrations of hydrogen peroxide in the workplace, ensure eyewash stations and safety showers are accessible, and use splash goggles, gloves, and an approved vapor respirator.

In the event of exposure to hydrogen peroxide, seek medical attention and follow these first aid guidelines:

• Inhalation— Seek fresh air. If victim’s breathing is difficult, administer oxygen. If breathing is absent, give artificial respiration and seek medical attention immediately.
• Eye Contact— Remove contact lenses if present. Immediately flush eyes with plenty of water for at least 15 minutes, and seek medical attention.
• Skin Contact— Flush skin with plenty of water and cover irritated skin with an emollient. Remove contaminated clothing. In case of serious skin exposure, use disinfectant soap and an anti-bacterial cream and seek medical attention.
• Ingestion— Do NOT induce vomiting. Loosen tight clothing. Never give anything by mouth to an unconscious person. Seek medical attention.

Storing & Disposing of Hydrogen Peroxide

Keep hydrogen peroxide away from sources of ignition, heat, and moisture, storing in a tightly closed container. Keep away from incompatible materials such as organic materials, metals, acids, alkalis, combustible materials, and oxidizing agents. This chemical must be disposed of in accordance with federal, state, and local environmental control regulations.

Need more information about hydrogen peroxide and other chemicals in your workplace? Browse our MSDS library to learn even more.

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Hydrogen Peroxide Experiments

The Effects of pH on Catechol Oxidase

Chemically, hydrogen peroxide has a similar composition to water, except its molecule has an additional oxygen atom. Simple experiments, some of which you can do at home, involve breaking down hydrogen peroxide into water and oxygen, using catalysts to quicken the reaction. Other experiments show the presence of oxygen. Hydrogen peroxide, in combination with other products, can produce visible chemical reactions.

TL;DR (Too Long; Didn't Read)

You can do simple experiments with drugstore hydrogen peroxide at home, breaking it down into water and oxygen.

Hydrogen Peroxide and Yeast

Hydrogen peroxide is relatively unstable, so over time it breaks down into water and oxygen. In this experiment, yeast is added to hydrogen peroxide to speed up its decomposition process, which is normally slow. You can perform the experiment at home in a sink. You'll need an empty large soda bottle, 3 percent hydrogen peroxide from a grocery store, one packet of active yeast, liquid dish soap and warm water. Mix about 113 grams (4 ounces) of the hydrogen peroxide with 56 grams (2 ounces) of dish soap in the soda bottle. Set aside and mix the packet of yeast with warm water, letting it sit for about five minutes. Pour the yeast mixture into the soda bottle. The reaction produces oxygen gas and the addition of liquid detergent creates foam.

Hydrogen Peroxide and Bleach

The mixture of hydrogen peroxide and bleach creates oxygen gas, salt (sodium chloride) and water. The bleach must contain sodium hypochlorite for this experiment to work. The solutions do not need to be concentrated to get a quick reaction. You will need 3 percent hydrogen peroxide, approximately 6 percent household bleach and a beaker. Pour 56 grams (2 ounces) of bleach into the beaker and the equivalent of hydrogen peroxide. Once the two are mixed, the reaction will occur quickly, producing bubbling.

Hydrogen Peroxide and Burning Sulfur

This experiment doesn't decompose hydrogen peroxide but merely shows that it contains oxygen. You expose a rose to burning sulfur and then dip it in hydrogen peroxide. You'll need two drinking cups, a rose with a small stem, tape, foil, sulfur and hydrogen peroxide. Tape the rose to the inside of the first cup and place a small pile of sulfur on a piece of aluminum foil. Add flame to the sulfur until it starts to smolder -- turn the cup with the rose upside down over the burning sulfur. The rose is exposed to sulfur dioxide gas, turning the petals of the rose to white as the gas combines with the oxygen in the colored part of the rose. Remove the rose from the cup and dip it into a cup filled halfway with hydrogen peroxide. The hydrogen peroxide provides oxygen to the flower, restoring its color.

Safety Considerations

Make sure to wear protective eyewear when conducting any of these experiments, whether at home or in a classroom or lab setting. If hydrogen peroxide comes in contact with your eyes, it can result in damage or blindness. It is imperative to seek medical attention if this happens. Make sure to wear an apron and clothing that covers your skin. According to the Agency for Toxic Substances and Disease Registry website, hydrogen peroxide can cause skin irritation -- there may also be skin burns with blisters with exposure to concentrated solutions. The peroxide you buy in the drug store is typically 3 percent, whereas chemists and other professionals might use stronger concentrations of 35 to 50 percent. Flush your skin with water if it is exposed to hydrogen peroxide.

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• Agency for Toxic Substances and Disease Registry: ToxFAQs for Hydrogen Peroxide
• Lansing Community College: The reaction of bleach and hydrogen peroxide

About the Author

Based in New Hamburg, Ontario, Mary Margaret Peralta has been writing for websites since 2010. She has developed a company website and a health and safety manual for a past employer. Peralta obtained her Bachelor of Arts in sociology from the University of Waterloo in Waterloo, Ontario.

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• Published: 13 September 2024

Perfluoroalkyl-modified covalent organic frameworks for continuous photocatalytic hydrogen peroxide synthesis and extraction in a biphasic fluid system

• Chaochen Shao 1 , 2 ,
• Xiaohan Yu 1 , 2 ,
• Yujin Ji   ORCID: orcid.org/0000-0003-3177-2073 1 , 2 ,
• Jie Xu   ORCID: orcid.org/0000-0002-4506-9701 3 ,
• Yuchen Yan 1 , 2 ,
• Yongpan Hu 1 , 2 ,
• Youyong Li   ORCID: orcid.org/0000-0002-5248-2756 1 , 2 ,
• Wei Huang   ORCID: orcid.org/0000-0003-3808-8258 1 , 2 &
• Yanguang Li   ORCID: orcid.org/0000-0003-0506-0451 1 , 2 , 4  

Nature Communications volume  15 , Article number:  8023 ( 2024 ) Cite this article

Metrics details

• Organic–inorganic nanostructures
• Photocatalysis

H 2 O 2 photosynthesis represents an appealing approach for sustainable and decentralized H 2 O 2 production. Unfortunately, current reactions are mostly carried out in laboratory-scale single-phase batch reactors, which have a limited H 2 O 2 production rate (<100 μmol h −1 ) and cannot operate in an uninterrupted manner. Herein, we propose continuous H 2 O 2 photosynthesis and extraction in a biphasic fluid system. A superhydrophobic covalent organic framework photocatalyst with perfluoroalkyl functionalization is rationally designed and prepared via the Schiff-base reaction. When applied in a home-built biphasic fluid photo-reactor, the superhydrophobicity of our photocatalyst allows its selective dispersion in the oil phase, while formed H 2 O 2 is spontaneously extracted to the water phase. Through optimizing reaction parameters, we achieve continuous H 2 O 2 photosynthesis and extraction with an unprecedented production rate of up to 968 μmol h −1 and tunable H 2 O 2 concentrations from 2.2 to 38.1 mM. As-obtained H 2 O 2 solution could satisfactorily meet the general demands of household disinfection and wastewater treatments.

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Introduction.

Hydrogen peroxide (H 2 O 2 ) is one of the most essential basic chemicals for synthetic industry, environmental remediation, and medical disinfection, with an annual global demand of 4.4 million tons and a market size of USD 3.2 billion in 2022 1 , 2 . During the recent COVID-19 pandemic, its demand has risen substantially owing to its wide use in the formulation of disinfectant products 3 . At present, over 95% of commercial H 2 O 2 is manufactured through the well-established anthraquinone oxidation process in centralized plants, involving substantial energy consumption and waste emission 4 . It generally yields highly concentrated H 2 O 2 , whose storage, transportation, and handling may pose significant safety risks. On many occasions, however, end-users only need dilute H 2 O 2 solution: for example, <0.1 wt% (or ~30 mM) H 2 O 2 is usually sufficient for water treatments and antibacterial purposes 5 , 6 . This notable gap between production and consumption stimulates us to search for alternative processes to enable the on-site, on-demand production of dilute H 2 O 2 solution 7 , 8 .

Solar-driven H 2 O 2 photosynthesis from oxygen and water has emerged as a promising route 9 , 10 , 11 . It uses solar energy as the sole energy input and generates no waste chemicals throughout the reaction process. Over recent years, a variety of photocatalysts have been developed with many exciting progresses 12 , 13 , 14 , 15 . They, unfortunately, have been predominantly investigated in laboratory-scale single-phase batch reactors, in which catalyst powders are dispersed in an O 2 -saturated aqueous solution (Fig.  1a ) 14 , 16 . While such a batch configuration is easy to operate and permits quick catalyst screening, its often limited size (solution volume <50 mL) and H 2 O 2 production rate (<100 μmol h −1 ) are far from amenable to practical applications 17 . Moreover, all the batch reactors only operate intermittently, and necessitate repetitive catalyst separation and recycling at intervals as short as a few hours to extract H 2 O 2 solution, resulting in low productivity and added expenses. Several attempts have been made to address this limitation. For example, biphasic batch reactors containing liquid water and oil phases have been shown to facilitate the spontaneous separation and collection of H 2 O 2 18 , 19 . Supporting photocatalyst powders on porous hydrophobic substrates floating on the solution surface creates abundant triple-phase boundaries, and promotes O 2 mass transfer and hence H 2 O 2 production 20 , 21 . Despite some performance gains, none of them could enable the continuous photosynthesis and extraction of H 2 O 2 at practically meaningful concentrations for directly connecting to the end users.

a Previously reported photocatalytic systems for H 2 O 2 production including the monophasic system, H 2 O-oil biphasic system, and gas-solid-liquid triphasic system. The colors yellow, blue, and pink represent the photocatalyst, water phase, and oil phase, respectively, while the gray grids denote the hydrophobic support. b Biphasic fluid system that enables continuous H 2 O 2 photosynthesis, separation, and extraction. The colors yellow, blue, and pink represent the photocatalyst, water phase, and oil phase, respectively.

We envision that a biphasic fluid system represents a promising solution to the above challenge (Fig.  1b ). Fluid reactors have the demonstrated potential for the continuous electrosynthesis or photosynthesis of a variety of valuable chemicals 22 , 23 , 24 , 25 , 26 . The introduction of biphasic water-oil reaction solution within fluid systems may benefit spontaneous H 2 O 2 separation while being continuously produced. To achieve so, desirable photocatalyst materials should have strong surface hydrophobicity in order to be stably and selectively dispersed in organic phases. Covalent organic frameworks (COFs)—a class of crystalline and porous polymer semiconductors—are appealing candidates by virtue of their versatile structural diversity, and tunable optoelectronic and surface properties 27 , 28 , 29 , 30 , 31 . Their interactions with solvents could, in principle, be modified by incorporating proper functional building blocks 32 . Based on the above reasoning, we here prepare a superhydrophobic COF photocatalyst via a judiciously designed Schiff-base reaction between tritopic amine and tetratopic aldehyde monomers. Their symmetry mismatch leaves periodical uncondensed aldehyde sites, which are subsequently grafted with perfluoroalkyl chains to afford the product with superhydrophobicity. This surface property allows the photocatalyst to be stably dispersed in the oil phase within a biphasic fluid photo-reactor. By properly adjusting reaction parameters, we achieve continuous production and extraction of pure H 2 O 2 solution with an exceptional production rate of up to 968 μmol h −1 and tunable H 2 O 2 concentrations from 2.2 to 38.1 mM. As-prepared H 2 O 2 solution can be used for household disinfection and environmental remediation.

Preparation and characterizations of BTTA-COF and PF-BTTA-COF

As schematically illustrated in Fig.  2a , our catalyst was prepared through a [4+3] Schiff-base condensation reaction between 5,5’-(benzo[ c ][1,2,5]thiadiazole-4,7-diyl)diisophthalaldehyde (BTDIPA) and 2,4,6-tris(4-aminophenyl)-1,3,5-triazine (TAPT) under a solvothermal condition (see the synthetic details in the  Supplementary Information ). The as-prepared sample from this step is denoted as BTTA-COF. Previous studies indicated that benzothiadiazole (BT) and triazine moieties favored the two-electron oxygen reduction reaction (2e − ORR) 33 , 34 , 35 . Their abundant incorporation into polymeric frameworks is expected to accelerate H 2 O 2 photosynthesis. Moreover, the symmetry mismatch between tritopic amines and tetratopic aldehydes leaves uncondensed aldehyde sites after the reaction, which could be subsequently functionalized with other molecules for regulating the photocatalyst surface wettability. Here in order to afford BTTA-COF with superhydrophobicity, its unreacted aldehyde sites were further condensed with 1H,1H-undecafluorohexylamine (UFHA) via the Schiff-base reaction. The final product is denoted as PF-BTTA-COF to reflect its perfluoroalkyl functionalization.

a Schematic synthetic procedure for BTTA-COF and PF-BTTA-COF. b FT-IR spectra of BTTA-COF, PF-BTTA-COF, and TAPT. c XRD patterns of BTTA-COF and PF-BTTA-COF. d TEM image of PF-BTTA-COF. e Water or α,α,α-trifluorotoluene (TFT) contact angle measurements of BTTA-COF and PF-BTTA-COF as well as the photograph showing their dispersion in biphasic H 2 O-TFT mixture. f Time-dependent H 2 O 2 evolution on BTTA-COF within batch reactors of two different sizes while retaining the same catalyst concentration.

The molecular structures of both samples were interrogated by spectroscopic characterizations. In the Fourier-transform infrared (FT-IR) spectrum of BTTA-COF, the emergence of the signature imine signal at 1627 cm −1 attests to the successful condensation between monomers (Supplementary Fig.  1a ) 36 . Its moderate signal at 1696 cm −1 indicates the existence of uncondensed aldehyde sites owing to the monomer symmetry mismatch as explained above, while no unreacted amine signal is noted in BTTA-COF (Fig.  2b ) 37 , 38 . The molar percentage of residual aldehydes is estimated to be 15%, close to the theoretical value (Supplementary Fig.  2a–c ). The subsequent introduction of perfluoroalkyl functionalities in PF-BTTA-COF does not disrupt the overall molecular structure (Supplementary Fig.  1b ). New peaks, however, are observed at 1237 cm −1 and 1201 cm −1 assignable to the C-F bonding in PF-BTTA-COF, and the signal intensity of uncondensed aldehydes significantly attenuates after modification, evidencing that perfluoroalkyl groups are successfully grafted to the polymeric frameworks 39 . Energy-dispersive X-ray spectroscopy (EDS) analysis reveals a fluorine content of 5.3 wt%, indicating that around one-third of unreacted aldehydes are modified by UFHA (Supplementary Fig.  2d, e ) 40 , 41 . The condensed molecular structure and incorporation of perfluoroalkane are also corroborated by the solid-state 13 C (Supplementary Fig.  3a ) and 19 F nuclear magnetic resonance (NMR) results (Supplementary Fig.  3b ).

Both BTTA-COF and PF-BTTA-COF feature great structural crystallinity. Their X-ray diffraction patterns (XRD) exhibit intense peaks that can be simulated by the eclipsed (AA) stacking of two-dimensional (2D) molecular layers (Fig.  2c and Supplementary Fig.  4 ). The strongest signal at 2θ = 6.1° corresponds to the (100) diffraction and evidences the in-plane ordering with a d -spacing of 14.4 Å—close to the pore-to-pore distance of the proposed structure (14 Å). N 2 sorption isotherms reveal their microporous nature (Supplementary Fig.  5a ). The Brunauer‐Emmett‐Teller (BET) specific surface area is calculated to be 1090 m 2 g −1 for BTTA-COF and 462 m 2 g −1 for PF-BTTA-COF, with an average pore size of 1.39 and 1.27 nm, respectively (Supplementary Fig.  5b ). The decreased surface area and pore size of PF-BTTA-COF result from its pore filling with perfluoroalkane as expected. Furthermore, scanning electron microscopy (SEM) imaging shows the rod-like morphology of both samples (Supplementary Fig.  6 ). High-resolution transmission electron microscopy (TEM) imaging unveils clear lattice fringes and supports their long-range structural ordering (Fig.  2d and Supplementary Fig.  7 ). Thermogravimetric analysis (TGA) under N 2 evidences the excellent thermal stability of both samples up to 500 °C (Supplementary Fig.  8 ).

Surface hydrophobicity is a prerequisite to the catalyst design for biphasic H 2 O 2 photosynthesis 18 . To investigate the effect of perfluoroalkyl functionalization, water contact angle (CA) measurements were conducted. As shown in Fig.  2e , unmodified BTTA-COF exhibits a static water CA of 31.4°, while the CA value is dramatically increased to 151.2° for PF-BTTA-COF. When α,α,α-trifluorotoluene (TFT)—a commonly used water-immiscible organic solvent—is dropped onto the surface of PF-BTTA-COF, the organic solvent quickly spreads and is absorbed in less than a second. These results reflect the superhydrophobicity and superoleophilicity of PF-BTTA-COF as a result of perfluoroalkyl functionalization. Thanks to this unique surface property, when its powder is added to an immiscible H 2 O-TFT mixture, PF-BTTA-COF immediately migrates to and becomes stably dispersed in the oil phase, forming a clear oil-water boundary. Such a feature is essential to the continuous H 2 O 2 production and extraction in our biphasic fluid system, as will be shown later. By sharp contrast, unmodified BTTA-COF does not form selective dispersion and can be suspended in both water and TFT.

We also examined the optoelectronic properties of our samples. The ultraviolet-visible (UV-Vis) diffuse reflectance spectrum of BTTA-COF displays an adsorption onset at 550 nm (Supplementary Fig.  9a ). This corresponds to an optical band gap ( E g ) of 2.58 eV according to the Tauc’s relation. Using Mott-Schottky and ultraviolet photoelectron spectroscopy (UPS) analyses, its conduction band (CB) and valence band (VB) positions are estimated to be −0.57 V and 2.01 V versus normal hydrogen electrode (NHE), respectively (Supplementary Fig.  9b, c ). The incorporation of perfluoroalkyl groups in PF-BTTA-COF does not noticeably modify the optoelectronic property (Supplementary Fig.  10a–c ). Based on their electronic structures, both samples are capable of driving simultaneous 2e − ORR and 4e − water oxidation reaction (4e − WOR) (Supplementary Figs.  9d and 10d ).

Photocatalytic measurements of BTTA-COF in a batch system

In order to evaluate the potential of our photocatalysts and to make a fair comparison with other competitors under similar conditions, we first conducted photocatalytic measurements of hydrophilic BTTA-COF in a conventional single-phase batch reactor. The catalyst powder was dispersed in 10 mL of pure water at an optimal concentration of 1 g L −1 (Supplementary Figs.  11 and 12 , see more photocatalysis details in the  Supplementary Information ). Under visible light irradiation ( λ  > 420 nm), BTTA-COF enables H 2 O 2 production and linear accumulation over time, yielding a total amount of 53 μmol after 2 h (Fig.  2f ). This corresponds to a H 2 O 2 production rate of 2650 μmol h −1 g −1 or a concentration accumulation rate of 2.65 mM h −1 . Control experiments show that H 2 O 2 is predominantly produced through the 2e − ORR by photogenerated electrons (Supplementary Fig.  13a ). This process involves the formation of both superoxide anion ( • O 2 − ) and endoperoxide (–O–O–) intermediates, as verified by electron paramagnetic resonance (EPR) and in-situ diffuse reflectance infrared Fourier-transform spectroscopy (DRIFTS) analyses (Supplementary Fig.  14 ), while photogenerated holes are responsible for driving the 4e − WOR to O 2 (Supplementary Fig.  13b ) 26 , 42 , 43 . Moreover, when benzyl alcohol (BA) is introduced as the sacrificial electron donor to accelerate hole consumption under otherwise identical conditions, the H 2 O 2 production rate and concentration accumulation rate are further boosted to 5691 μmol h −1 g −1 and 5.69 mM h −1 , respectively (Supplementary Fig.  15 ). The apparent quantum efficiency (AQE) at 420 nm is measured to be 18% in pure water (Supplementary Fig.  16a ) and 37.4% in the presence of BA (Supplementary Fig.  16b ). In addition, the solar-to-chemical energy conversion (SCC) efficiency is calculated to be 0.48% in pure water (Supplementary Fig.  17 ). All these metrics are comparable to other state-of-the-art candidates in pure water or aqueous solutions involving sacrificial electron donors under similar conditions (Supplementary Table  1 ) 19 , 26 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 .

BTTA-COF has the required stability for H 2 O 2 photosynthesis in pure water. It shows great compatibility with H 2 O 2 , and does not catalyze H 2 O 2 degradation even under visible light irradiation (Supplementary Fig.  18 ). The cycling photocatalytic experiment demonstrates a negligible activity loss after 6 reaction cycles and a total of 30 h (Supplementary Fig.  19 ). Characterizations of the catalyst retrieved after the cycling experiment reveal no discernable change in its structure or optoelectronic properties (Supplementary Fig.  20 ). Note that PF-BTTA-COF exhibits a comparable photocatalytic activity to BTTA-COF under identical reaction conditions, which is expected given their similar optoelectronic properties (Supplementary Fig.  21 ).

Despite their wide use in photocatalytic measurements, single-phase batch reactors are not amenable to practical applications owing to their limited solution volumes and intermittent operation. We attempted to address the first issue by enlarging the batch reactor and increasing the solution volume to 150 mL (15 times larger) while maintaining the same catalyst concentration and light illumination intensity. Unfortunately, the H 2 O 2 yield does not proportionally increase: the total amount only increases 3 times in pure water, while the normalized H 2 O 2 production rate and concentration accumulation rate are substantially lowered to 0.4 mM h −1 and 400 μmol h −1 g −1 , respectively (Fig.  2f ). This leads us to conclude that we cannot simply scale up the reaction in batch reactors owing to insufficient light penetration depth and catalyst utilization in large sized reactors 50 . More importantly, batch reactors are not suitable for continuous H 2 O 2 production and extraction, which we aim for in this study.

Continuous H 2 O 2 photosynthesis and extraction by PF-BTTA-COF in a biphasic fluid system

A biphasic fluid system was developed to address the unavoidable drawbacks of conventional batch reactors. It consists of three main components, as depicted by the photo in Fig.  3a . O 2 -saturated photocatalyst dispersion in an organic solvent and pure water are co-fed into a T-shaped mixer through peristaltic pumps at controlled flow rates. This generates a series of stable oil-water biphasic segments inside transparent tubular flow channels, which are subsequently transported into a coiled reactor (total channel length ~44 m) irradiated by a set of light-emitting diodes ( λ  = 455 nm, 25 mW cm −2 ). During the reaction, the small diameter (~1.6 mm) of flow channels reduces the light penetration path when side-irradiated; the presence of abundant biphasic oil-water interfaces promotes H 2 O 2 extraction from the oil phase to the water phase (Fig.  3b ). The oil/water segment lengths and density of the biphasic interfaces could be readily tuned by varying the relative liquid feeding rates (Fig.  3c ). After passing through the coiled reactor, the H 2 O 2 solution and photocatalyst dispersion are fed to a collector, where they spontaneously separate into two layers of liquids due to their immiscibility (Supplementary Fig.  22 ). The upper H 2 O 2 solution is extracted for practical applications, while the lower photocatalyst dispersion is pumped back for subsequent use. Such a biphasic fluid system can continuously yield pure H 2 O 2 solution with tunable concentrations.

a Photographs of the home-built biphasic fluid photocatalytic system in operation. Dashed squares highlight (i) the T-shape mixer for mixing water and oil flows and (ii) spontaneous product separation and collection. b Schematic illustration of H 2 O 2 formation in TFT and migration across the oil-water interfaces. c Typical images of oil-water segments formed inside tubular flow channels at different flow rates and F W /F O ratios. d H 2 O 2 production rate and average solution concentration from the biphasic fluid system at different flow rates (F W  = F O ). Error bars represent the standard deviations of three independent experiments. e H 2 O 2 production rate and average solution concentration at different F W /F O ratios (F W  + F O  = 2.12 mL min −1 ). Error bars represent the standard deviations of three independent experiments. f Performance comparison of PF-BTTA-COF in our biphasic fluid system with those of other state-of-the-art photocatalysts in terms of H 2 O 2 production rate and concentration. Open and filled circles represent the studies in pure water and in the presence of sacrificial electron donors, respectively.

We used superhydrophobic PF-BTTA-COF in the above-developed biphasic fluid system for continuous H 2 O 2 production as a proof of concept. TFT was chosen as the organic solvent in our study considering its excellent wetting of PF-BTTA-COF, complete immiscibility with water, and high oxygen solubility 51 , 52 . Our catalyst powder was dispersed in TFT at a concentration of 2 g L −1 . When the feeding rates of water (F W ) and TFT (F O ) are both set at 0.43 mL min −1 to start with, the catalyst retention time inside the coiled reactor is about 100 min, and our biphasic fluid system continuously produces pure H 2 O 2 solution at a rate of 99 μmol h −1 (Supplementary Fig.  23 ). The introduction of BA in TFT as the sacrificial electron donor further enhances the H 2 O 2 production rate to 318 μmol h −1 and yields H 2 O 2 solution at a concentration of 12.2 mM. Note that BA and its oxidation product benzaldehyde have much higher solubility in TFT than in water, as evidenced by the 1 H NMR analysis (Supplementary Fig.  24 ). This is essential for biphasic H 2 O 2 photosynthesis. As a result, our following optimization is approached in the presence of BA.

We examined the effect of liquid flow rates on photocatalytic activities. Increasing the flow rates is expected to reduce the catalyst retention time in the coiled reactor, and therefore decrease the attainable H 2 O 2 concentration. For example, when both F W and F O are increased to 0.71 mL min −1 , the H 2 O 2 concentration is lowered to 9.1 mM; it is further lowered to 6.1 or 3.1 mM at F W  = F O  = 1.08 or 2.02 mL min −1 , respectively (Fig.  3d ). Varying the overall flow rate while keeping F W  = F O does not significantly change the H 2 O 2 production rate (300 ~ 400 μmol h −1 ). This is because equal F W and F O values always result in equi-length water and oil segments in tubular flow channels and thereby similar light utilization efficiency. We also investigated the effect of the F W /F O ratio while keeping the same overall flow rate (2.12 mL min −1 ). At a large F W /F O ratio of 2, the H 2 O 2 production rate is measured to be 363 μmol h −1 , and the H 2 O 2 concentration is 4.3 mM (Fig.  3e ). Both values improve with decreasing F W /F O ratios due to the enhanced light utilization efficiency by the catalyst dispersed in oil. At the lowest F W /F O ratio of 1/4 (as limited by our peristaltic pumps), the H 2 O 2 production rate and H 2 O 2 concentration are measured to be 616 μmol h −1 and 24.3 mM, respectively. Furthermore, lowering the pH value of the water phase with dilute acid is found to promote H 2 O 2 photosynthesis. For example, the H 2 O 2 production rate of PF-BTTA-COF is boosted to 847 μmol h −1 at pH = 3 and the optimal flow rates, while alkaline pH adversely affects the performance owing to spontaneous H 2 O 2 decomposition under the alkaline condition (Supplementary Fig.  25a ) 53 . At last, rising the BA content in TFT to 50 vol% further enhances the H 2 O 2 production rate and concentration to 968 μmol h −1 and 38.1 mM (0.13 wt%) respectively under the optimal flow rates and water pH (Supplementary Fig.  25b ). To our best knowledge, such extraordinary H 2 O 2 production rate is 1 ~ 2 order of magnitude greater than all earlier studies (10 ~ 100 μmol h −1 ) (Fig.  3f and Supplementary Table  1 ), thereby unambiguously underlining the unique advantage of our biphasic fluid system 34 , 35 , 45 , 48 , 49 , 54 , 55 , 56 , 57 , 58 , 59 .

We next carried out continuous H 2 O 2 photosynthesis using our biphasic fluid system at F W  = 1.04 mL min −1 and F O  = 2.08 mL min −1 . As shown in Fig.  4a , the total H 2 O 2 amount linearly accumulates at the first 80 h, and the increment slightly slows down as the reaction proceeds. After 100 h, more than 35 mmol of H 2 O 2 is produced with an average H 2 O 2 production rate of 357 μmol h −1 , eventually giving rise to more than 6 L of H 2 O 2 solution with a concentration of 5.7 mM (Fig.  4b ). The collected liquid product can be directly used for multiple applications. Using two bacteria Staphylococcus aureus ( S. aureus ) and Escherichia coli ( E. coli ), as examples, we find that their growth is fully inhibited with the application of 5.7 mM H 2 O 2 solution (Fig.  4c and Supplementary Fig.  26 ). Our product solution can also be employed in wastewater treatment as simulated by the Fenton reaction in the presence of methyl blue (MB) or methyl orange (MO) at practically relevant concentrations (100 ppm) 60 . Both organic dyes are observed to totally degrade within 30 s after the introduction of 5.7 mM H 2 O 2 solution (Fig.  4d, e and Supplementary Fig.  27 ). The above results showcase that the dilute H 2 O 2 solution produced from our biphasic fluid system can satisfactorily meet the demands of household disinfection and environmental remediation. The solution could be stored for over 1 month without significant degradation (Supplementary Fig.  28 ).

a Total amount of H 2 O 2 produced and its concentration change over time during an uninterrupted 100 h test in our biphasic fluid system. b Photographs of the as-obtained H 2 O 2 solution. c Antibacterial tests against S. aureus and E. coli using the as-obtained H 2 O 2 solution. d , e Degradation of d MB and e MO using the as-obtained H 2 O 2 solution, insets are the photographs showing the dye decolorization.

To evaluate the economic feasibility of our biphasic fluid system, we carried out a techno-economic analysis (TEA) based on an amplified reactor device to determine the levelized cost of the product (LCP) and the end-of-life net present value (NPV) 61 , 62 . The estimation of the capital and operational costs is based on the prevailing market price of raw materials and products, as summarized in Supplementary Tables  2 and 3 . It is found that when the F W /F O ratio is set at 1/4, LCP is most economical (Supplementary Fig.  29a ). Assuming a facility lifespan of 10 years, the end-of-life NPV turns profitable by the fourth year (Supplementary Fig.  29b ). These results demonstrate the practical viability of the biphasic fluid system in H 2 O 2 production.

In summary, we here demonstrated an innovative strategy for continuous H 2 O 2 photosynthesis and extraction. The success key lies in the judicious design of both the photocatalyst material and the reactor. PF-BTTA-COF was prepared via the Schiff-base reaction between tritopic amine and tetratopic aldehyde monomers, followed by perfluoroalkyl functionalization at uncondensed aldehyde sites to afford its surface superhydrophobicity. Such a surface property allows the photocatalyst to be selectively and stably dispersed in oil instead of water. We then constructed a biphasic fluid system by co-feeding water and O 2 -saturated catalyst-dispersed TFT through transparent tubular flow channels to a coiled reactor under irradiation. The immiscibility of these two liquid phases led to the formation of a series of stable TFT-water biphasic segments with clear interfaces that promoted H 2 O 2 spontaneous extraction. By properly adjusting reaction conditions, we achieved an unprecedented H 2 O 2 production rate of up to 968 μmol h −1 and tunable H 2 O 2 concentrations from 2.2 to 38.1 mM. As-obtained H 2 O 2 solution could be directly supplied to end-users where and when it is needed, and can satisfactorily meet the practical requirements of disinfection and wastewater treatments. Moreover, our biphasic fluid system could be readily scaled up by increasing the channel length or connecting several coiled reactors in parallel or in series.

Synthesis of BTTA-COF and PF-BTTA-COF

Typically, BTDIPA (30.0 mg, 0.075 mmol) and 2,4,6-tris(4-aminophenyl)-1,3,5-triazine (TAPT) (26.6 mg, 0.075 mmol) were ultrasonically dispersed in a mixture of o -dichlorobenzene, n -butyl alcohol, and 6 M acetic acid (4.4 mL, 5/5/1, v/v/v) in a 25 mL Pyrex tube. The tube was degassed by three vacuum-N 2 filling cycles, sealed under vacuum, and heated at 120 °C for 72 h. After cooled down to room temperature, the solid was collected by centrifugation, thoroughly washed with N , N -dimethylformamide (commercial sources and purities), anhydrous tetrahydrofuran, and acetone, respectively, and finally dried under vacuum at 80 °C overnight to afford BTTA-COF as a light-yellow powder (yield: 88%). For the synthesis of PF-BTTA-COF, BTTA-COF (100 mg) was ultrasonically dispersed in ethanol (5 mL), then added with UFHA (500 µL, 2.5 mmol) and acetic acid (50 µL) under stirring. The mixture was stirred at room temperature for 5 h under N 2 . The product was isolated by filtration, thoroughly washed with ethanol, and dried under vacuum at 80 °C overnight.

Determination of H 2 O 2 concentration

The H 2 O 2 concentration was determined using a colorimetric method as described in our previous publication 34 . Typically, a ferrous ion oxidation xylenol orange (FOX) solution was prepared by dissolving Fe(NH 4 ) 2 (SO 4 ) 2 ·6H 2 O (19.61 mg), D-sorbitol (3.644 mg), and xylenol orange (XO) (14.333 mg) in deionized water (200 mL) added with ethanol (2 mL) and H 2 SO 4 (98%, 272 μL). Subsequently, 50 μL of the obtained H 2 O 2 solution (diluted if needed) was mixed with the pre-prepared FOX solution. The concentration of H 2 O 2 was quantified by monitoring the characteristic absorption peak at 550 nm via UV-Vis spectroscopy according to the calibration curve (Supplementary Fig.  11 ).

Photocatalytic H 2 O 2 production in a batch system

In a typical batch photocatalytic reaction, 10 mg of photocatalyst was dispersed in 10 mL of pure water (or with 10 vol% BA) inside a top-irradiated Pyrex reactor (120 mL). The suspension was first bubbled with O 2 for 30 min before the reactor was carefully sealed. During the photocatalytic reaction, the reactor was irradiated by a 300 W Xe-lamp (China Education Au-light, CEL-HXF300) with a cutoff filter of 420 nm. The light intensity was calibrated to be 200 mW cm −2 using a Newport light-power meter (Model 1918-R). To monitor the reaction process, the reaction solution was extracted every 1 h and filtrated through a syringe filter (0.22 μm) to remove the photocatalyst powder. The resulting H 2 O 2 concentration was quantified using the FOX solution.

Photocatalytic H 2 O 2 production in the biphasic fluid system

In a typical photocatalytic reaction, 140 mg of PF-BTTA-COF photocatalyst was ultrasonically dispersed in 70 mL of TFT (with or without 10 vol% BA). The TFT dispersion (oil phase) and distilled water (water phase) were separately bubbled with O 2 for 30 min, and then pumped through a T-shape valve at specific feeding rates by two peristaltic pumps and mixed together. This led to the formation of consecutive oil-water segments, which were fed into a coiled tubular reactor made of polypropylene tubing ( Φ 1.6 × 3.2 mm). The total tube length in the reactor is 44 m, corresponding to a total volume of ~90 mL. The solution retention time in the flow channel was controlled by the overall flow rate of water and oil. Inside the coiled reactor, the oil-water segments were side-irradiated by a set of LED arrays with a wavelength of 455 nm and a light intensity of 25 mW cm −2 . During the photocatalytic reaction, generated H 2 O 2 would migrate across abundant oil-water interfaces and accumulate in the water phase. After passing through the coiled reactor, the oil-water mixed solution was collected in a container, where phase separation occurred spontaneously due to the immiscibility of water and TFT. The upper H 2 O 2 aqueous solution was directly extracted using a peristaltic pump for subsequent uses, while the lower photocatalyst dispersion was pumped back for the next reaction cycle. Such a system achieved continuous H 2 O 2 production and extraction as well as photocatalyst recycling. The H 2 O 2 concentration at the outlet was determined every 1 h using the FOX solution.

Long-term continuous H 2 O 2 photosynthesis in the biphasic system

The long-term continuous photocatalysis in the biphasic fluid system was conducted under similar conditions. 1.2 g of PF-BTTA-COF was dispersed in 300 mL of O 2 -saturated TFT and BA (30 mL, 9:1, v/v). The flow rates of oil and water phases were kept at 1.04 mL min −1 and 2.08 mL min −1 , respectively. The H 2 O 2 concentration at the outlet was determined every 2 h using the FOX solution.

Antibacterial experiments

The antibacterial effect of the as-obtained H 2 O 2 solution was estimated by the plate colony counting method. Staphylococcus aureus ( S. aureus ) and Escherichia coli ( E. coli ) were selected as the target bacteria. An individual colony was first cultured in a fresh LB agar plate by shaking at a speed of 200 rpm at 37 °C for 12 h. Then, 30 µL of the initial colony solution was subsequently diluted 100-fold and shaken for another 3 h at 37 °C to ensure bacterial growth in the log-phase, eventually resulting in ~10 6 colony-forming units (CFU) per milliliter. Then 100 µL of the bacterial solution was incubated with the as-obtained H 2 O 2 solution (500 µL, 5.7 mM) for 3 h. The colonies were photographed after 24 h incubation at 37 °C. Control experiments were conducted under identical conditions except that H 2 O 2 solution was not added.

Dye degradation experiments

Methyl blue (MB) and methyl orange (MO) were chosen as representative organic dyes for the degradation experiments using the Fenton reaction process. In a typical experiment, a stock solution containing dye (100 ppm) and FeSO 4 (6 mM) was prepared, and its pH value was adjusted to 3.0 using diluted H 2 SO 4 . Subsequently, 2 mL of the as-obtained H 2 O 2 solution (5.7 mM) was added to 2 mL of the above-mentioned dye solution in the dark. The absorbance of the organic dye at its maximum absorption wavelength was recorded using UV-Vis spectroscopy, and its concentration was calculated based on the calibration curve with standard solutions.

Data availability

All the data that support the findings of this study are provided in the  Supplementary Information . Other data are available from the corresponding author upon request.  Source data are provided with this paper.

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Acknowledgements

We acknowledge the financial support from National Natural Science Foundation of China (U2002213 and 52161160331), the Natural Science Foundation of Jiangsu Province (BK20220027), the Science and Technology Development Fund Macau SAR (0077/2021/A2), the Natural Science Foundation of the Jiangsu Higher Education Institutions of China (20KJA430002), and the Collaborative Innovation Center of Suzhou Nano Science and Technology. We thank Zihui Han and Prof. Liang Cheng for the antibacterial tests. We thank Yuhang Wang for techno-economic analysis.

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Chaochen Shao, Xiaohan Yu, Yujin Ji, Yuchen Yan, Yongpan Hu, Youyong Li, Wei Huang & Yanguang Li

Jiangsu Key Laboratory of Advanced Negative Carbon Technologies, Soochow University, 215123, Suzhou, China

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W.H. and Y.G.L. conceived the project and designed the experiments. C.S. synthesized the catalysts and performed the structural characterizations and photocatalytic tests. C.S. and X.Y. carried out the long-time performance evaluation. Y.J. and Y.Y.L. conducted the structural simulations. J.X. and Y.Y. performed the HR-TEM imaging and EDS mapping. Y.H. assisted on the quantification of H 2 O 2 . C.S., W.H., and Y.G.L. co-wrote the manuscript with contributions from all co-authors.

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Shao, C., Yu, X., Ji, Y. et al. Perfluoroalkyl-modified covalent organic frameworks for continuous photocatalytic hydrogen peroxide synthesis and extraction in a biphasic fluid system. Nat Commun 15 , 8023 (2024). https://doi.org/10.1038/s41467-024-52405-3

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Alternating red-blue light alleviated physiological injury by reducing oxidative stress under both high light and continuous light from red-blue LEDs

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• Wenke Liu   ORCID: orcid.org/0000-0003-4433-8735 1 , 2 &
• Jiayuan Liu 1 , 2  

CL (continuous light)-sensitive lettuce cultivar were treated under high light (HL, 480 µmol·m − 2 ·s − 1 , 20/4 h) and CL (400 µmol·m − 2 ·s − 1 , 24/0 h) provided by red-blue LEDs (red-blue ratio 3:1) without or with R/B alternation (HL-A and CL-A, alternating duration ratio 3:1) at the same daily light integral. On the 16th day after light treatment, lettuce was diurnally sampled four times at equal 6 h intervals, thus the effects of HL-A and CL-A on growth, leafy injury, yield, and carbohydrate accumulation, oxidative stress and their circadian rhythms of lettuce were investigated and evaluated. The results showed that the lettuce plants under HL-A and CL-A had greater canopy size, leafy area and shoot fresh biomass than those under HL and CL correspondingly. However, the specific leaf weight, SPAD value of new leaves and leaf injury grade were decreased. The contents of starch and sucrose, and DPPH free radical scavenging rate were significantly increased under HL-A and CL-A compared with HL and CL, while the contents of hydrogen peroxide, superoxide anion and MDA were decreased. Compared with HL and HL-A, the above physiological indices of lettuce under CL and CL-A were higher respectively. HL-A and CL-A did not change the circadian rhythm of lettuce. In conclusion, HL-A and CL-A improved the yield and external quality of CL-sensitive lettuce. Importantly, they alleviated HL- and CL-induced physiological injuries of lettuces by reducing the accumulation of ROS only, rather than decreasing carbohydrate accumulation or circadian rhythm disorder.

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This research was financially supported by the National Natural Science Foundation of China (NSFC) (No. 31672202).

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Liu, W., Liu, J. Alternating red-blue light alleviated physiological injury by reducing oxidative stress under both high light and continuous light from red-blue LEDs. Hortic. Environ. Biotechnol. (2024). https://doi.org/10.1007/s13580-024-00611-9

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Non-lethal exposure to H 2 O 2 boosts bacterial survival and evolvability against oxidative stress

Alexandro rodríguez-rojas.

1 Freie Universität Berlin, Institute of Biology, Evolutionary Biology, Berlin, Germany

Joshua Jay Kim

Paul r. johnston.

2 Leibniz-Institute of Freshwater Ecology and Inland Fisheries (IGB), Berlin, Germany

3 Berlin Center for Genomics in Biodiversity Research, Berlin, Germany

Olga Makarova

Murat eravci.

4 Freie Universität Berlin, Institute of Chemistry and Biochemistry, Berlin, Germany

Christoph Weise

Regine hengge.

5 Humboldt-Universität zu Berlin, Institut für Biologie/Mikrobiologie, Berlin, Germany

Associated Data

All relevant data are within the manuscript and its supporting Information files, including raw data. All sequencing data can be retried from the following repository: https://www.ncbi.nlm.nih.gov/bioproject/PRJNA485867/

Unicellular organisms have the prevalent challenge to survive under oxidative stress of reactive oxygen species (ROS) such as hydrogen peroxide (H 2 O 2 ). ROS are present as by-products of photosynthesis and aerobic respiration. These reactive species are even employed by multicellular organisms as potent weapons against microbes. Although bacterial defences against lethal and sub-lethal oxidative stress have been studied in model bacteria, the role of fluctuating H 2 O 2 concentrations remains unexplored. It is known that sub-lethal exposure of Escherichia coli to H 2 O 2 results in enhanced survival upon subsequent exposure. Here we investigate the priming response to H 2 O 2 at physiological concentrations. The basis and the duration of the response (memory) were also determined by time-lapse quantitative proteomics. We found that a low level of H 2 O 2 induced several scavenging enzymes showing a long half-life, subsequently protecting cells from future exposure. We then asked if the phenotypic resistance against H 2 O 2 alters the evolution of resistance against oxygen stress. Experimental evolution of H 2 O 2 resistance revealed faster evolution and higher levels of resistance in primed cells. Several mutations were found to be associated with resistance in evolved populations affecting different loci but, counterintuitively, none of them was directly associated with scavenging systems. Our results have important implications for host colonisation and infections where microbes often encounter reactive oxygen species in gradients.

Author summary

Throughout evolution, bacteria were exposed to reactive oxygen species and evolved the ability to scavenge toxic oxygen radicals. Furthermore, multicellular organisms evolved the ability to produce such oxygen species directed against pathogens. Recent studies also suggest that ROS such as H 2 O 2 play an important role during host gut colonisation by its microbiota. Traditionally, experiments with different antimicrobials have been carried out using fixed concentrations while in nature, including in intra-host environments, microbes are more likely to experience this type of stress in steps or gradients. Here we show that bacteria treated with sub-lethal concentrations of H 2 O 2 (priming) survive far better than non-treated cells when they subsequently encounter a higher concentration. We also found that the 'priming' response has a protective role from lethal mutagenesis. This protection is provided by long-lived proteins that, upon priming, remain at a high level for several generations as determined by time-lapse LC-mass spectrometry. Bacteria that were primed evolved H 2 O 2 resistance faster and to a higher level. Moreover, mutations that increase resistance to H 2 O 2 , as determined by whole-genome sequencing (WGS), do not occur in known scavenger encoding genes but in loci coding for other functions, at least in E . coli .

Introduction

The ability to elicit a stress response when encountering repeated stress relies on 'remembering' a similar event from the past (memory), a trait common to many biological entities [ 1 ]. During the course of an infection or the colonisation of a host, bacteria encounter increasing and repeated stress imposed by the host immune system [ 2 , 3 ]. Obata et al ., for example, recently demonstrated that low levels of H 2 O 2 in the gut of Drosophila melanogaster shape the composition of the gut microbiota resulting in differential survival of the flies [ 3 ]. Here, we report how bacteria respond to the exposure to low levels of reactive oxygen species (ROS) and how this impacts bacterial fitness. We then investigate if the phenotypic response to a sub-lethal dose of H 2 O 2 facilitates resistance evolution, hence providing a test of the plasticity-first hypothesis, which proposes that environmentally initiated phenotypic change precedes or even facilitates evolutionary adaptation [ 2 , 4 ].

In Escherichia coli , the defences against oxidative stress depend on transcriptional regulators such as OxyR or SoxR that detect changes in the redox balance. They also induce the production of detoxifying enzymes, DNA repair and protection systems and other proteins [ 5 ]. Oxidation of OxyR by H 2 O 2 leads to the formation of an intramolecular disulfide bond between cysteine residues 199 and 208. Oxidized OxyR positively regulates catalases and peroxidases. OxyR is deactivated by enzymatic reduction with glutaredoxin I (Grx) or thioredoxin (Trx). Because the Grx/GorA system is itself transcriptionally regulated by OxyR, the whole response is self-regulated in a homeostatic feedback loop [ 6 ].

The OxyR-mediated oxidative stress response results in scavenging of H 2 O 2 and mitigates the toxicity of this by-product of aerobic metabolism. It includes the induction of Suf proteins that form a complex to supply apo-enzymes with iron-sulfur clusters. The Suf system replaces the normal iron-sulfur cluster supply system (Isc), required for critical biochemical pathways such as respiration, which is disrupted by H 2 O 2 stress [ 7 , 8 ]. The iron-sulphur clusters of dehydratases are one of the most H 2 O 2 -sensitive systems. The repair of those clusters by Suf is necessary to prevent the failure of the TCA cycle. The iron-sulfur clusters from enzymes that employ ferrous iron as a co-factor can increase the risk of fuelling a Fenton reaction. Active OxyR also induces Dps, a ferritin-class protein, that strongly suppresses the amount of DNA damage by sequestering the unincorporated iron [ 9 ]. OxyR mutants accumulate ROS at much higher levels than the wild-type strain during growth even in the absence of H 2 O 2 which also accounts for its high sensitivity [ 10 ].

The spontaneous reaction of H 2 O 2 with free ferrous iron (Fe 2+ ) at physiological pH, oxidising iron to Fe 3+ and generating hydroxyl radicals and water, is named the Fenton reaction. Hydroxyl is a strong non-selective radical that damages many cellular components, particularly DNA [ 11 , 12 ]. H 2 O 2 impedes the function of the Fur regulatory protein and can directly damage many cell components but is less toxic than other reactive oxygen species such as hydroxyl. These radicals are responsible for DNA damage, indirectly promoted by H 2 O 2 as a consequence of the Fenton reaction [ 13 ].

A classic paper by Imlay et al . describes that the pre-treatment of E . coli with a low dose (60 μM) of H 2 O 2 can increase the survival upon subsequent exposure to an otherwise lethal dose (30 mM) [ 14 ]. However, neither the duration of such priming responses nor the molecular mechanisms of its maintenance, i.e. the memory, have been studied. Our study has two main aims. First, we investigated the main factors in the H 2 O 2 priming response and for how long the response is sustained. Second, we used this system to test the hypothesis that inducible phenotypes accelerate adaptive evolution [ 4 ]. Therefore, we experimentally evolved Escherichia coli under increasing concentrations of H 2 O 2 with and without priming. For H 2 O 2 -resistant populations evolved after priming and non-priming regimes, genome re-sequencing analyses were performed to identify mutations. We focus on growing bacteria (exponential phase), as this better represents infection or colonisation of host surfaces including the gut [ 2 , 3 ]. Moreover, this allows us to focus on the priming response in proliferating cells, where the response to H 2 O 2 differs from that in stationary phase. While the response to the H 2 O 2 in exponentially growing bacteria is mostly controlled by OxyR, RpoS, the master regulator of the general stress response controls a pronounced phenotypic H 2 O 2 tolerance during stationary phase [ 15 , 16 ].

Results and discussion

Priming by h 2 o 2 results in higher bacterial survival.

We found that, in our conditions, the minimal inhibitory concentration for H 2 O 2 is 1 mM. This concentration was subsequently used as a reference for 30-minute killing curves that show a clear dose-effect in survival rate ( Fig 1A , ranging from 50 μM to 1 mM, Fig 1A , p = 2.1x10 -16 , DRC model fitted based on maximum likelihood [ 17 ]). Based on these results, we primed cells for 30 min with 0.1 mM H 2 O 2 and challenge the cultures with 1 mM H 2 O 2 90 min after priming stimulus. The priming concentration (0.1 mM) is the maximum dose that shows no difference in growth rate for each time-point compared to non-treated cells ( S1 Fig , S1 Table ). Primed (pre-treated) populations of E . coli showed higher survival than naïve cells by more than one order of magnitude (Cox proportional hazard model, p <0.05, Fig 1B ). We also determined that the priming response contributes to a more efficient removal of H 2 O 2 by quantifying it in the supernatant of the cultures ( S2 Table ). After 15 minutes, H 2 O 2 dropped from 1 mM to 0.723 mM for control while in pre-treated cultures H 2 O 2 decreased to 0.140 mM ( p = 0.00014, pre-treated versus control). For 30 minutes, the level of H 2 O 2 went down to 0.377 mM for control cultures while in pre-treated cultures, it decreased to 0.0222 mM ( p = 0.00035).

Panel A shows bacterial sensitivity to 1 mM H 2 O 2 with a strong dose-response effect ( p = 2.1x10 -16 , DRC model fitted based on maximum likelihood). Panel B shows the priming effect or improved response when cells are treated in advance with a non-lethal dose (0.1 mM). Curves represent the mean of five independent cultures per time point. Asterisks represent significant differences (Welch’s test, one asterisk for p <0.05 and two asterisks for p <0.01). Only comparisons between primed and non-primed groups are shown.

Although there is no difference in growth rate, priming with 0.1 mM H 2 O 2 imposes a small cost (approximately 4%, S1 Fig , S1 Table ). The cost can be detected by comparing the empirical area under the curve (p = 0.0002, 0.9619 fold-change) or the carrying capacity of the curve ( p = 0.0001, 0.9565 fold-change) from primed bacteria culture versus non-treated control by allowing bacteria to grow until the stationary phase. No other parameter such as initial population size, growth rate, or doubling time is affected. Therefore, differential survival can be attributed only to cell response but not to cell growth arrest. The cost can be explained by even very low concentrations of H 2 O 2 damaging the iron-sulfur clusters, thereby compromising respiration. However, at low bacteria density in rich medium, E . coli can grow very fast by fermentation [ 18 ]. This wasteful strategy allows quick generation of ATP at the expense of the medium and can explain the small difference in carrying capacity and the area under the curve of the growth curves ( S1 Fig , S1 Table ). In our experiments, this cost has no consequences because bacteria are maintained in low density and in exponential growth. This scenario should be similar while starting an infection or colonisation of a host's gut.

These results are in agreement with data previously reported by Imlay et al . [ 11 , 14 ], showing survival protection even to doses as high as 30 mM, a concentration close to H 2 O 2 usage as a disinfectant. Our concentrations are also in the range of some in vivo situations. For example, tailfin transection on zebrafish larvae induces a rapid increase in H 2 O 2 levels ranging from 100–200 μM in the wound margins [ 2 ]. In some cases, more than 100 μM of H 2 O 2 have been reported in human and animal eye vitreous humour and aqueous humour [ 19 ]. In plants, the average tissue concentration of H 2 O 2 is around 1 mM, but under stress it can as high as 10 mM [ 20 ]. This could be relevant for foodborne pathogens transmitted by consumption of contaminated vegetables.

Duration of the priming response

As the priming response protects the cells effectively from an otherwise lethal exposure to H 2 O 2 , an important question is for how long this response remains effective. To address this, we pre-treated E . coli again with 0.1 mM H 2 O 2 for 30 minutes, but we applied the higher dose (1 mM, trigger of the priming response) at different time-points (30, 60, 90, 120 and 150 minutes after the 30 min priming period, cell density kept constant by appropriate dilution). We observed a significant decay of the priming response from 120 minutes after H 2 O 2 pre-treatment removal ( Fig 2 ), approximately four divisions, suggesting that the priming effect is also trans-generational. After 150 minutes, the survival rate of primed populations no longer differed from naïve populations. Another study has shown long-term memory based on an epigenetic switch that controls a bimodal virulence alternation also in the scale of hours in E . coli [ 21 ].

During memory decline experiment, the viability of the cells remains unaltered before the addition of the trigger (A). The priming memory declined over time after induction of priming response with 0.1 mM H 2 O 2 (trigger) (B). Asterisks indicate significant differences between each time-point pair (Welch’s test, one asterisk for p <0.05 and two asterisks for p <0.01).

How is the state of priming maintained for up to five generations? Bacteria can store information about recent stress via stable transcripts or proteins [ 21 , 22 ]. We investigated the memory at the protein level and initially studied the impact of priming and trigger concentrations (0.1 and 1 mM) on the proteome of E . coli by quantitative LC-mass spectrometry. We detected upregulation of many of the known enzymes that are induced by H 2 O 2 just 5 minutes after the addition of H 2 O 2 ( Fig 3 , and S3 Table and S4 Table ). For both concentrations used, we detected and quantified many proteins belonging to the OxyR regulon. Furthermore, many other genes, such as ahpC / F , xthA and suf operons showed a weak difference or no response at all. The samples in this experiment were taken after only 5 minutes of treatment. The rationale behind this design is that the availability of viable cells after 1 mM H 2 O 2 treatment would be too low at a later stage. At a concentration of 0.1 mM, however, it is possible to collect viable cells for any other time-point after exposure. These results are presented in Fig 4 .

Hierarchical clustering of the intensities was performed using Euclidean distances between means. Rows indicate the fluctuation of protein (by gene names) level at 5 minutes after addition of 0.1 and 1 mM H 2 O 2 . Intensity ranges of the log2 fold-changes are given from highest intensity (green) to lowest (red). Only the 50 most statistically significant up-regulated proteins are shown, taking as a reference the 0.1 mM concentration.

Hierarchical clustering of the intensities was performed by using Euclidean distances between means. Rows indicate the fluctuation of protein (by gene names) level at different time points. Intensity ranges of the log2 fold-changes are given from highest intensity (green) to lowest (red). Only the most 50 statistically significant up-regulated proteins at time point T1 (30 minutes after priming) are represented to follow their fluctuation in the other time points with 30 minutes between them.

To explore the temporal dynamics of the proteome after a 30-minute stimulus with H 2 O 2 (0.1 mM), we followed the changes over almost 3 hours (five time points: 30, 60, 90, 120 and 150 minutes). The decline in anti-H 2 O 2 protein levels correlates well with the decrease of the response ( Fig 4 ). Proteins such as KatG, AhpF or RecA declined slowly after removal of the H 2 O 2 , consistent with a sustained production with a minor contribution of dilution due to cell division and slow degradation rate. These results indicate that many of these proteins are stable and show a significantly higher abundance than in the control even at 150 minutes after H 2 O 2 treatment. Other initially induced proteins such as GrxA, YaaA and XthA, SufA, SufS, AcrA-AcrB had completely declined at this point indicating that these proteins may be subject to proteolysis and have shorter half-lives ( Fig 4 ) but also that the stressful situation is alleviated. The overall results of this proteomic experiment show that the memory of the priming response in E . coli is mediated by the scavenging proteins such as KatG and AhpCF. The primary amino-acid sequence is informative about the in vivo half-life a protein. An N-end rule-based prediction of the half-life for some of the proteins that are responsible for the memory additionally is consistent with our findings obtained by the proteomic approach ( S5 Table ).

We visualised the global impact of H 2 O 2 on bacterial physiology using a network analysis based on protein-protein interactions and function [ 23 ]. This network analysis provides information on protein level alterations. It integrates protein-protein interactions, including indirect (functional) and direct (physical) associations [ 23 ]. We have projected our proteomic datasets over the established interactions of E . coli proteins to illustrate the scope of the priming response, including toxic effects. The proteome response to the priming concentration (0.1 mM H 2 O 2 during 30 minutes), resulted in a high degree of connectivity of protein-protein interactions and functional relation of both, up- and down-regulated proteins ( Fig 5 ). If we compare our network with a large-scale protein-protein interaction network of E . coli [ 24 ], we find a wide perturbation including the most important nodes. This analysis also points to proteome-wide readjustments to cope with H 2 O 2 stress and shows the profound impact of oxidative stress across the entire proteome even at a low dose that does not change the growth rate in a rich medium. The damage induced to iron-sulfur clusters even at a low H 2 O 2 concentration (0.1 mM) might have no detectable impact on bacterial cell growth in early exponential phase, since under these conditions most of the energy is obtained by fermentation, as shown for E . coli previously [ 18 ].

Pale green nodes indicate up-regulated proteins while pale red ones represent down-regulated ones. Note that the most critical nodes (DnaK and GloL) of the protein-protein interaction network are affected. The interaction among nodes shows the proteome-wide impact of H 2 O 2 .

This means that at low cell density in rich medium, the consumption of the resources does not drastically change the medium properties and it is advantageous for bacteria to use a costly strategy that provides them with fast energy via fermentation which supports faster growth compared to aerobic respiration.

The memory could also be based on long-lived transcripts. Therefore, we sequenced the full transcriptome after exposure to H 2 O 2 . RNAseq captured both sRNA and mRNA and we sampled just before the decline of the response (120 minutes after removal the stimulus). The transcript with the greatest induction was OxyS, a small regulatory RNA (sRNA) induced by active OxyR ( S6 Table and S7 Table ). OxyS regulates several genes, but although several targets have been identified, its function is not fully understood [ 25 ]. We did not detect upregulation of other transcripts under OxyR regulation.

OxyS is a potential candidate to explain the duration of priming because it is relatively stable with a half-life between 18 to 20 minutes during the exponential phase [ 25 – 27 ]. To explore this possibility, we used an oxyS- deficient mutant to test its sensitivity to H 2 O 2 over 30 minutes. We did not find any significant differences in sensitivity ( S2 Fig ) consistent with prior reports [ 26 – 28 ]. Although OxyS did not provide us with a mechanism to explain the duration of the memory, its stability could play some role in alleviating DNA damage as recently suggested [ 29 ]. However, such protection does not seem to have a significant impact on cell survival.

Based on the comparison of proteomic and transcriptomic data it seems reasonable to assume that the capacity of the cells ‘to remember’ the stimulus is mainly based on the stability of scavenging proteins as documented in the proteomic dataset. These scavenging proteins remain present at higher concentrations than in the control samples as long as 120 minutes after removal of peroxide. This indicates that these enzymes are not degraded but probably their production continues after the stimulus during several cell divisions, which could explain the pattern that we observe in the decline of the response. It is important to consider that around 120 minutes, there were still significantly higher levels of KatG and AhpF than at the start of the experiment. Thus, the memory may require the contributions of additional genes whose relative expression decreased to low levels after two hours. It is also possible that specific enzymatic activity of KatG and AhpF gets lost over time, since the proteomic approach is based on protein identification by sequence, not by activity.

Priming response is compromised by disrupting important H 2 O 2 -scavenging genes

The expression of genes important for H 2 O 2 stress survival was also confirmed by qPCR 30 minutes after the addition of the chemical (0.1 mM). We found a significant up-regulation of selected genes such as katG , ahpF / ahpC , dps , mntH and sufA ( S4 Fig and S8 Table ). As previously described in the literature, oxyR and fur do not change in expression level when cells are treated with H 2 O 2, since their activation relies on switching between active or inactive forms of these proteins which depends on the intracellular level of H 2 O 2 or iron respectively [ 6 , 7 ]. Many of these transcripts showed a strong induction at the RNA level ( S8 Table ). After 30 minutes, the level of induction of all genes showed a stronger response at the RNA level than at the protein level. In a normal situation, we would expect that a single molecule of RNA is translated into several proteins. This possibly indicates that under oxidative stress, the translation is inefficient and it is compensated by a high level of transcription probably due to damage of many cell components, including ribosomes as previously described [ 30 ].

To understand the priming response to H 2 O 2 at the molecular level, we constructed a set of mutants for genes encoding key proteins identified in our proteomic dataset. In proliferating E . coli , OxyR is the major regulator controlling the cellular response to H 2 O 2 [ 10 ]. We explored the involvement of OxyR in the priming response since many of the differentially expressed proteins were transcribed in an OxyR-dependent fashion.

We found that by disrupting OxyR, there was a dramatic change in sensitivity to H 2 O 2 with full loss of viability after 30 minutes ( S2 Fig ). The priming response is completely abolished ( Fig 6 ), indicating that the enhanced survival, due to pre-exposure to H 2 O 2 , depends on the regulator OxyR. This regulator is a major transcription factor that protects E . coli against H 2 O 2 during the exponential phase [ 10 , 31 ]. The active form of OxyR positively regulates dozens of genes.

Asterisks represent significant differences (Welch’s test, one asterisk for p <0.05 and two asterisks for p <0.01). Only comparison between primed and non-primed groups are shown.

Next, from the most highly expressed proteins and informed by published work [ 6 , 10 ], we generated a set of mutants that were used to determine the contribution of the respective genes to survival ( S2 Fig ) and priming ( Fig 6 ). Removal of KatG (catalase), AhpF (one subunit of the alkyl peroxidase AhpCF) or RecA (DNA repair) significantly decreased the survival in the presence of H 2 O 2 (1 mM). The double mutant for KatG/AhpF showed even stronger sensitivity. However, the absence of other important proteins induced by H 2 O 2 such as YaaA (decreases intracellular iron [ 31 ]), LipA (lipoate synthase, important for the repair of iron-sulfur clusters [ 32 , 33 ]), GltD (glutamate synthase subunit), GhxA or GhrA did not result in differential survival at 1 mM concentration. The OxyS sRNA was the transcript with the highest level of induction in the RNAseq data, but the mutant did not display any increased sensitivity to H 2 O 2 , it neither suppresses or decreases the priming response compared to the wild-type control. Assuming that H 2 O 2 sensitivity and the priming response are closely linked, we included in the additional priming response experiment mainly those mutants with increased sensitivity ( Fig 6 and S2 Fig ). The removal of KatG indicates that catalase importantly contributes to the priming response, but it does not fully explain the protection observed for the WT strain (compare to Fig 1B ). In the absence of AhpF, we also observe differences in priming response with the naïve state but not as pronounced as in the case of KatG-deficient strain. The double mutant defective in KatG and AhpF showed a dramatic decrease in the priming response but was still significantly different from naïve cells. Recently, a report showed that bacteria lacking the AhpF/C system suffer a severe post-stress recovery [ 34 ]. The absence of the AhpF/C system also contributes to the lethality of the mutants probably by the inability of the cell to cope with a low level of ROS after severe oxidative stress [ 34 ]. Another protein that illustrates an important influence on priming response is RecA, with the mutant showing a decreased priming response to H 2 O 2 . Overall, our data indicate that the priming by a low dose of H 2 O 2 is multifactorial, with several OxyR-controlled proteins such as KatG, AhpCF or other factors related to DNA as RecA contributing to the priming response.

Priming response enhances the survival of evolving populations

To find out whether the priming response described above has an influence on the rate of evolution of resistance to H 2 O 2 , we used an experimental evolution approach. We evolved bacterial cultures with a treatment protocol described in detail in the M&M section. Parallel populations were evolved where one group was periodically exposed to a sub-lethal concentration of H 2 O 2 , an activating signal that should protect the populations in comparison with naïve ones when later exposed to a higher dose. We continued daily intermittent exposures to H 2 O 2 doubling both, priming and triggering concentrations until extinction occurred. We consider that a population is extinct when it shows no sign of growth during the next passage. This was also confirmed by periodical contamination checks after each passage. We observed that the extinction rate was faster for the naïve populations in comparison to primed populations (Log-rank test, p < 0.05, Fig 7 ). Naïve cells evolved resistance allowing them to resist up to 8 mM H 2 O 2 , whereas primed cells evolved resistance to survive up to 16 mM H 2 O 2 . These results show that priming increases the evolvability of pre-treated populations. We repeated the selection experiment including an additional control to exclude the possibility that primed populations evolved better because they received 10% more H 2 O 2 (priming plus triggering concentrations together). The result of this experiment showed a similar pattern to the previous one, naive populations went extinct first, including the one receiving ten percent more H 2 O 2. Both naive populations groups were not significantly different ( S3 Fig ).

Note that every passage was carried out every 24 hours although time in the x-axis is represented as continuous. Both, priming and triggering doses were increased twofold daily up to 32 mM, where total extinction occurred. The extinction was perceived by negative growth in the next passage and by the absence of growth in LB plates during contamination controls. Non-evolving population control (grey line, 20 populations) is also shown. Evolvability differs between the two treatments, naïve (red line) and primed (blue line) populations (Log-rank test, p < 0.01). Differences with non-evolved populations were not determined.

Further analysis of the resistant populations by whole-genome sequencing revealed that all populations harboured different sets of mutations. Surprisingly, we did not find any direct modification in the enzymatic scavenger systems, such as catalase or any other proteins related to peroxide protection, but we cannot exclude changes in expression level of these systems due to regulatory mutations acting in trans . There are hundreds of single nucleotide polymorphisms (SNPs) and other types of mutations that were unique to each population for both regimes ( Fig 8 ). Many of these mutations probably represent neutral or non-lethal changes that populations accumulated during the exposure to H 2 O 2 . Here, H 2 O 2 is not only a selective agent, but it also speeds up evolution by increasing mutagenesis. At the moment, we cannot be certain about the contribution of particular mutations to H 2 O 2 resistance and they will be subject to detailed studies in the future. However, we studied two cases of the most frequent mutations in more detail as a proof of principle.

The positions indicate the approximate locations of mutations in the E . coli MG1655 chromosome relative to the wild-type. Different types of mutations that were found in both evolving regimes are indicated in the left panel highlighted with different colours (non-synonymous amino acid changes in yellow, intergenic mutations in green and frameshift in red). The right panel indicates mutations that were present for both regimes (grey) and those that were exclusive to the priming regime (blue). To see full reports of mutation for each population see supplementary material.

One first case is that of very frequent inactivating mutations in fimE . The promoter of the fimbrial subunit gene, fimA (the first gene of the operon fimAICDFGH ) lies within a short segment of invertible DNA known as the fim switch ( fimS ), and the orientation of the switch in the chromosome determines whether fimA is transcribed or not. Inversion is catalysed by two site-specific recombinases, the FimB and FimE proteins. The FimB protein inverts the switch in either direction, while FimE inverts it predominantly to the off orientation. When FimB and FimE are co-expressed, FimE activity dominates and the switch turns to the off phase, wherease a fimE knockout mutation increases fimbriae production [ 35 ].

We selected one of the many fimE defective mutants (Δ1 bp, position 248 out of 597 nucleotides) for the next experiments. This type of mutant was only present in primed populations although there were some other intergenic mutations between fimE→fimA , both in primed and non-primed populations , pointing to the relevance of this type of mutation in survival under H 2 O 2 stress . A series of experiments showed that fimE mutants attach to glass surfaces more efficiently than the wild-type strain, suggesting that production of fimbriae is active in the mutant. Furthermore, the fimE mutant showed decreased susceptibility to H 2 O 2 , with an MIC of 4 mM in compared to 1 mM in the parental strain. The transformation of the mutant strain with a plasmid overexpressing fimE (pCA24N- fimE , GFP minus) [ 36 ] reverted the attaching ability ( Fig 9 ) of the mutant and also restored the original resistance to 1 mM H 2 O 2 . In early biofilm research it was shown that type I fimbriae (the product of the fim operon) are required for submerged biofilm formation. Type I pili (harbouring the mannose‐specific adhesin, FimH) are required for initial surface attachment [ 37 ]. Fimbriae expression per se constitutes a signal transduction mechanism that affects several unrelated genes via the thiol-disulfide status of OxyR [ 38 ]. Fimbriae formation is accompanied by massive disulfide bridge formation [ 38 ] that could also contribute to titration of the exogenous H 2 O 2 , thereby limiting the intracellular damage.

The image was taken after vital staining (LIVE/DEAD BacLight Bacterial Viability Kit). Top panel shows greater attachment and reduced sensitivity to H 2 O 2 MIC associated with fimE inactivation. Complementation restores both H 2 O 2 sensitivity and the low-attachment phenotype of the wild-type strain (lower panel). In both panels control cells (transformed with the cloning vector pCA24N) can be also observed.

Following the inversion of the phase switch to ‘on state’ of fimbriae production by environmental signals, this element can remain phase-locked in the ‘on orientation’ due to integration of insertion sequence elements at various positions of the fimE gene [ 39 ]. Interestingly, fim operon expression allows E . coli to attach to abiotic surfaces, host tissues and to survive better inside macrophages protecting against the presence of extracellular antibacterial compounds [ 40 , 41 ]. Reactive oxygen species (ROS) are critical components of the antimicrobial repertoire of macrophages to kill bacteria [ 42 ].

A second case that we studied is the intergenic mutations between the genes insB1 and flhD (insB1→flhD) , which occurred at high frequency in our evolution experiment. FlhD is coexpressed from an operon with FlhC and both proteins form the master transcriptional factor that regulates transcription of several flagellar and non-flagellar operons by binding to their promoter regions [ 43 ]. It is known that in E . coli MG1655 , some insertion sequences such as insB1 can increase the motility of E . coli [ 44 ]. Our hypothesis here was that mutations in the intergenic region between insB1 and flhD contribute to abolishing the positive effect of insB1 insertions on motility, which in turn increases resistance to H 2 O 2 .

We first analysed whether mutants from our evolution experiment showing these mutations showed decreased motility. Two independent mutants (positions 1978493 nt, Δ10bp and 1978504 nt, Δ1 bp) from two different populations showed decreased swimming and increased sedimentation when culture tubes were not shaken, as was the case in the evolution experiment setup. Both mutants also showed a MIC of 4 mM to H 2 O 2 , compared to 1mM in the control. The transformation with a plasmid overexpressing the operon flhDC (pVN15) restored both motility and H 2 O 2 sensitivity ( Fig 10 ). These mutations may protect against H 2 O 2 through the mechanism of decreased motility, which results in clustering of the cells at the bottom of the culture, which may improve protection of cells located inside the clusters.

Panel A shows the initial phenotypes of insB1→flhD mutants , low motility and resistance to 4 mM H 2 O 2 while the parental strain is motile with a MIC of 4 mM H 2 O 2 . A complementation experiment showed identical phenotypes when the strains were transformed with the cloning vector (panel B) and recovery of motility and original resistance to 1 mM H 2 O 2 when both mutants are transformed with the plasmid overexpressing the operon flhDC [ 80 ] (panel C).

A second possibility is that the decrease of motility itself decreases the basal level of H 2 O 2 due to lower metabolic demand. Flagellar motility enables bacteria to escape from detrimental conditions and to reach more favourable environments [ 44 ]. However, flagella impose an important energetic burden on bacterial metabolism due to the number of proteins involved in the machinery as well as the associated energy expenditure associated with motility. For instance, one interesting study showed improved tolerance to oxidative stress in Pseudomonas putida as reflected by an increased NADPH/NADP(+) ratio, concluding that flagellar motility represents the archetypal trade-off involved in acquiring environmental advantages at the cost of a considerable metabolic burden [ 45 ]. In our condition of oxidative stress, the flagellate phenotype makes the cells more susceptible to H 2 O 2 . These results raise an interesting question in regard to the motile vs non-motile strategy in bacteria: does flagellar activity bring diminishing returns by creating sensitivity to oxidative stress? The decreased expression in the flagellar gene hierarchy also affects pdeH , a class III gene in this hierarchy, which encodes the major c-di-GMP degrading enzyme in E . coli . The result is an increase in c-di-GMP levels, which promotes the production of curli fibers, which are a major component of the extracellular biofilm matrix. The strain MG1655 does not produce cellulose; strains that do so would have also increased cellulose production. Curli fibers and cellulose production are co-regulated by CsgD, which is itself under positive c-di-GMP control. Thus, the reduction on FlhDC level promotes biofilm formation, which contributes to multiple stress resistance, including resistance against H 2 O 2 [ 46 ].

We can speculate about the role of some other mutations. For example, a set of changes are located in genes coding for iron-binding proteins or related to iron transport such as iceT , feoA , yaaX / yaaA or rsxC . The control of intracellular iron is crucial to decrease the adverse effects of Fenton chemistry [ 7 ]. There were also mutations that were common to both types of population, evolved under priming and non-priming conditions ( Fig 8 ). The most frequent mutations were yodB (a cytochrome b561 homologue), intergenic mutations between insA and uspC (universal stress protein C). Another frequent mutation was in the gene yagH , belonging to the CP4-6 prophage. CP4-6 is a cryptic prophage in E . coli that could play a role in bacterial survival under adverse environmental conditions [ 47 ].

Priming alters the mutational spectrum and H 2 O 2 -induced mutagenesis in evolving populations

To assess if the evolved populations have a similar mutational spectrum, we analysed the total pool of mutations segregated by the treatments. We used the Monte Carlo hypergeometric test implemented by iMARS [ 48 ] to assess the overall differences between each mutational spectrum. Both groups, evolved under priming and non-priming conditions, differed from each other significantly ( p = 0.00021). ROS induces a particular type of mutations, with a characteristic signature in the DNA. The guanine base in genomic DNA is highly susceptible to oxidative stress due to its low oxidation potential. Therefore, G·C→T·A and G·C→C·G transversion mutations frequently occur under oxidative conditions [ 49 , 50 ]. Thus, we investigated the proportion of C→A and C→G substitutions between the two types of evolving regimes, but we did not find significant differences ( p = 0.056 and p = 0.11 respectively, two-tailed Fisher’s exact-test, Fig 11 ).

Mutational spectra. The number of mutations (nucleotide substitutions and indels) is plotted against respective nucleotide positions within the gene fragment. In this analysis, the mutations were taken by type regardless of the targets by generating two frequency datasets to compare frequency by type of mutation of evolved populations under priming and non-priming regimes. The mutational spectra are significantly different between the two conditions according to the spectrum analysis software iMARS [ 46 ].

A possible explanation for these results is that the pre-activation of antioxidant defences helps to decrease the number of non-adaptive mutations and lethality by decreasing the extent to which the Fenton reaction occurs and the amount of released hydroxyl radicals. We observed an increase in the frequency of substitutions G→A, T→C, G→C, T→G and A→G in naïve populations compared to primed populations, indicating that the priming response greatly buffers DNA-damage. Primed populations showed an increased frequency of frameshift mutations by deletion of 1 bp, and additions of C or G that were only present in primed populations that also showed a higher number of A→C substitutions.

We hypothesised that the priming response would decrease DNA damage and hence also the rate of lethal mutations. To assess this possibility, we tested if priming can decrease the mutant frequency and hence lethality due to mutagenesis. Applying the conditions of the previous experiments, we determined the mutation frequency with and without H 2 O 2 treatment. We found that the priming response decreased H 2 O 2 -induced mutation frequency close to one order of magnitude compared to naïve cells ( p <0.001 primed versus naïve, Welch’s test, S5 Fig ). In addition, there is a very small (1.87-fold change) but significant difference between primed bacteria and the basal E . coli mutagenesis ( p = 0.034 primed versus base level, Welch’s test), indicating that priming suppresses most of the mutagenesis caused by H 2 O 2 . In our experiment, naïve and primed populations showed different death rates ( Fig 1 ). We also showed previously that different inoculum sizes in similar conditions to our current experiment do not influence mutagenesis [ 51 ]. We propose that one of the most important consequences of the priming response to H 2 O 2 is a drastic decrease in lethal mutagenesis. Although H 2 O 2 damages most of the cellular components [ 52 ], DNA damage is likely the major contributor to lethality.

In principle, an increase in mutation rate increases the evolvability of asexual populations [ 53 – 55 ]. How is it possible that naïve populations show lower evolvability compared to primed populations despite a higher mutation rate? A possible explanation is that evolvability can be influenced by the population size and the mutation supply. Even with increased mutagenesis, if population size drastically decreases, the final number of mutants can be smaller when survival is improved. We also found that the mutational spectra of our evolving populations are different. Mutational spectra are a qualitative property of mutation rate that could enhance or hinder the access to beneficial mutations [ 56 ]. It is possible that the observed changes in mutational spectra between primed and naïve evolving populations could play a role in balancing the ratio of deleterious/beneficial mutations, although demonstrating this will be the subject of future research.

Conclusions

Priming by H 2 O 2 in exponentially growing bacteria is quantitatively driven by genes that are mostly under OxyR control, with other genes such as recA also contributing to survival. The memory of the priming response can last up to four generations upon first exposure to H 2 O 2 . We also showed high stability of H 2 O 2 -detoxifying proteins, which play a significant role in resistance.

How general priming is and to which other stressors it applies remains to be seen. Our findings are also contributing to an understanding of ROS-mediated interactions of hosts with pathogens and the microbiota.

Our finding that priming boosts evolvability of bacterial populations by enhancing survival under oxidative stress, provides evidence for the phenotype as a target of selection during evolutionary processes and supplies a validation for the ‘plasticity-first’ hypothesis [ 4 ].

If such selection happens in the more complex environment of a host remains to be studied. Bacteria, however, are certainly frequently exposed to fluctuating concentration of ROS, concentrations that often will be sublethal.

The type and number of mutations indicate that scavenger systems against oxidative stress are optimally evolved since no mutations directly affecting these systems were found under H 2 O 2 stress selective pressure. Our results suggest that the ubiquitous occurrence of H 2 O 2 has an impact on bacterial lifestyle and the evolution and regulation of flagella motility. Moreover, mutations that result in bacterial clustering or increase bacterial density can contribute to protection against H 2 O 2 as shown by mutations of the flagella regulator or in the fim operon. It seems possible that H 2 O 2 thereby stimulates the early stages of biofilm formation, thus providing additional protection against ROS.

Materials and methods

Bacteria and growth conditions.

E . coli MG1655 was used as bacterial model for all experiments with H 2 O 2 . For genetic manipulation, Escherichia coli strain DH5α was used and routinely cultured in Lysogeny Broth (LB medium), supplemented with antibiotics when appropriate. All bacterial strains were cultured in Lysogeny Broth Lenox (Carl Roth, Germany). For all experiments with H 2 O 2 the LB was freshly prepared and kept in the dark until use.

Construction and verification of deletion mutants

All mutants used in this work ( S9 Table ) were generated in E . coli K‐12 strain MG1655 following a modified methodology described elsewhere [ 57 ]. Briefly, transformants carrying the red recombinase helper plasmid, pKD46, were grown in 5-ml SOB medium with ampicillin (100 μg/ml) and L-arabinose at 30°C to an OD 600 of ~0.5 and then made electrocompetent. PCR products with homology regions were generated using specific primers ( S10 Table ) to amplify the region of interest from the corresponding mutants of the Keio collection [ 58 ]. The PCR-generated fragments were purified (MinElute PCR Purification Kit, Qiagen). Competent cells in 50 μl aliquots were electroporated with 100 ng of PCR product. Cells were added immediately to 0.9 ml of SOC, incubated 1 h at 37°C, and then 100 μl aliquots spread onto LB agar with kanamycin (30 μg/ml). The mutants were verified by PCR and the antibiotic resistance cassette was removed using the plasmid pCP20. The correct inactivation of genes was verified by PCR. To construct double mutants, single mutants obtained in MG1655 were transduced using the P1vir phage procedure as previously described [ 59 ].

Hydrogen peroxide susceptibility testing

We determined the minimal inhibitory concentration for H 2 O 2 by broth microdilution method with some modifications. We used LB medium instead of Mueller-Hinton Broth and we used approximately 10 7 bacteria instead of 10 5 , a bacterial density that corresponds to the subsequent experiments. When working with H 2 O 2 , we carried out all experiments with freshly-made LB to avoid the accumulation of chemically formed H 2 O 2 by the joint action of light and Flavin [ 60 ] present in the medium during storage. Time-kill curves to H 2 O 2 were determined by exposing exponential phase bacteria at a density of ~ 2x10 7 CFU/ml to different concentrations and times, taking as a reference the modified MIC value.

Priming experiments with H 2 O 2

Starting from 2x10 7 CFU/ml, E . coli was exposed (stimulus) to 0.1 mM H 2 O 2 during 30 minutes at 37°C with shaking. The H 2 O 2 was removed by centrifugation at 4 000 x g for 10 minutes and cells were allowed to recover for 90 minutes, keeping the cell density constant by removing the required volume and replacing it with the appropriate amount of fresh pre-warmed-LB (37°C) every 30 minutes. The trigger (1 mM H 2 O 2 ) was added 90 minutes after removal of the stimulus. The challenge lasted for 30 minutes. At this point, 4 μg/ml catalase (Sigma Aldrich, Germany) was added to each tube and cultures were diluted and plated to determine cell viability. Non-treated cells were used as control. Each group consisted of five cultures.

Determination of the priming cost

Bacterial growth curves were measured in flat-bottom 96-well micro-plates (Nunc, Denmark). 40 independent colonies from a LB agar plate were inoculated in a 96 multi-well plate containing 200 μl/well of LB and incubated overnight at 37 °C in a humid chamber to prevent evaporation. In a new plate, each well was filled with 100 μl of fresh-made LB and inoculated per duplicate with 1 μl from the overnight plate (80 wells in total). The remaining 16 wells were used as medium contamination control. The plate was incubated for 2.5 hours to reach an OD 600 of 0.4. Then, 90 μl of medium per well were removed and replaced with 80 μl of fresh LB using a multichannel pipette. Ten additional microliters containing 1 mM H 2 O 2 were added to every 40 wells and mixed immediately. Only LB was added to the remaining 40 control wells. Each independent colony was represented in both experimental groups. The plate was placed into a microplate reader Synergy H1 (Biotek, Germany) and kinetic readings (OD 600 ) were carried out every 20 minutes after short shaking of 5 seconds. The cost of priming by H 2 O 2 (0.1 mM) was estimated from the parameters of the growth curves. All model parameters—carrying capacity, initial population size, growth rate, doubling time and the empirical area under the curve—were calculated using Growthcurver R package [ 61 ]. Each parameter for E . coli MG1655 growing with or without 0.1 mM H 2 O 2 (priming concentration), was compared using Student’s t-test.

Hydrogen peroxide colourimetric quantitation in E . coli supernatant media

Pierce Quantitative Peroxide Assay Kit (Thermo Scientific, Germany) was used according to manufacturer’s instructions. Briefly, E . coli cells were grown in fresh LB (Lennox) medium to OD 595 0.5. The cultures were diluted 10 times in 1 ml of fresh LB containing 0.1 mM H 2 O 2 (final concentration, primed) or LB alone (naïve). The tubes were incubated with moderate shaking for 30 minutes. Then, the supernatant was removed by centrifugation at 10000 x g during 1 minute and aspiration. The pellets were washed once with LB and resuspended in 1 ml of LB containing 1 mM H 2 O 2 . At time points 0, 15 and 30 minute 100 μl of supernatant were removed to determine the H 2 O 2 concentration. Finally, 20 μl microliters of medium supernatant per tube were diluted 10 times and mixed in a 96-well microplate with 200 μl of working solution (prepared according to the manufacturer’s instructions). The mix was incubated at room temperature for 15 minutes in a humid chamber. OD 600 was measured using a Synergy H1 plate reader (Biotek, Germany) and a standard curve of H 2 O 2 was prepared as indicated in the protocol. The blank value (working without H 2 O 2 ) was automatically subtracted from all sample measurements. Three independent cultures were used per group and non-treated cultures were used as an additional control. Means and standard deviations were calculated and compared for each time-point by a Welch's test.

Memory of the priming response

After applying a stimulus (0.1 mM H 2 O 2 ) for 30 minutes, the cells were allowed to recover for 30, 60, 90, 120 and 150 minutes before the addition of the trigger concentration (1 mM H 2 O 2 ). During the experiment, the OD 600 of all cultures were kept around 0.05 by adding suitable volume and removal of equivalent quantity to maintain the population in the exponential phase and the same population size. Bacteria remained exposed to the trigger for 30 minutes before starting the dilution in 1 ml of LB containing 4 μg of catalase. Appropriate dilutions were made and plated onto LB agar to determine the survival rate.

Global proteomics by LC-mass spectrometry

E . coli strain MG1655 was grown at 37°C in LB medium to an OD 600 of 0.5. The cultures were diluted 10 times in fresh LB. H 2 O 2 was added to tubes to final concentrations of 0.1 and 1 mM. Non-treated samples were used as control. All tubes were incubated for 5 minutes with shaking at 37°C. Remaining H 2 O 2 was removed by adding 4 μg/ml of catalase first and by centrifugation at 10 000 x g during 2 minutes. After removal of the supernatant, the equivalent amount of fresh LB was added. For the memory decline experiment, the procedure was identical, except that only 0.1 mM H 2 O 2 was used and the samples were treated for 30 minutes. After removal of the treatment, samples were taken after 30, 60, 90, 120 and 150 minutes. Each experimental condition consisted of six replicates. One millilitre per sample was pelleted by centrifugation at 10 000 x g for 2 minutes. The cells were resuspended in 50 μl of TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA) containing chicken lysozyme (0.1 mg/ml, Sigma Aldrich, Germany) and incubated at room temperature for 5 minutes with occasional swirling. A volume of 250 μl of denaturation buffer (6M urea/2 M thiourea in 10 mM HEPES pH 8.0) was added into each sample and 25 μl (which approximately corresponds to 50 μg of protein) of the resulting lysate were used for in-solution protein digestion as described previously [ 62 ]. Briefly, proteins, re-suspended in denaturation buffer, were reduced by the addition of 1 μl of 10 mM DTT dissolved in 50 mM ammonium bicarbonate (ABC) and incubated for 30 minutes, followed by 20-minute alkylation reaction with 1 μl of 55 mM iodoacetamide. As a first digestion step, Lysyl endopeptidase (LysC, Wako, Japan) resuspended in 50 mM ABC was added to each tube in a ratio of 1 μg per 50 μg of total proteins and incubated for 3 hours. After pre-digestion with LysC, protein samples were diluted four times with 50 mM ABC and subjected to overnight trypsin digestion using 1 μg/reaction of sequencing grade modified trypsin (Promega, USA), also diluted before use in 50 mM ABC. All in-solution protein digestion steps were performed at room temperature. After the addition of iodoacetamide, the samples were protected from the light until the digestion was stopped by acidification adding 5% acetonitrile and 0.3% trifluoroacetic acid (final concentrations). The samples were micro-purified and concentrated using the Stage-tip protocol described elsewhere [ 62 ], and the eluates were vacuum-dried. Re-dissolved samples were loaded on a ReprosilPur C18 reverse phase column and peptides were analysed using a nano-HPLC Dionex Ultimate 3000 system (Thermo Scientific, Germany) coupled to an Orbitrap Velos mass spectrometer (Thermo Scientific, Germany). MS and MS/MS data from each LC/MS run were analysed with MaxQuant software [ 63 ]. Identification of proteins was performed using the MaxQuant implemented Andromeda peptide search engine and statistical analysis was carried out using the software Perseus [ 64 ].

Prediction of in vivo protein half-life and stability index

The half-life estimation is a prediction of the time that it takes for half of the amount of protein in a cell to disappear after its synthesis. It relies on the "N-end rule" (for a review see [ 65 – 67 ]). The instability index provides an estimate of the stability of a protein in a test tube. Based on experimental data [ 68 ], making possible to compute an instability index using the amino-acid sequence. For these predictions, we used the online software ProtParam [ 69 ]. When available, N-end sequence was corrected to the real in vivo sequence due to methionine excision [ 70 ].

Quantification of gene expression

To quantify the expression of H 2 O 2 stress related genes in E . coli , bacteria treated with 0.1 mM H 2 O 2 were compared to an untreated control. Preparation of each of the six treatments and the control samples were carried out by two operators three times on three consecutive days, thereby comprising three biological replicates for each of the six experimental and one control groups. Overnight (ON) cultures were diluted 1:100 in fresh LB, subdivided into six 10 ml aliquots and incubated in 50 ml-Falcon tubes at 37°C for 2.5 hours at 220 R.P.M. When the OD 600 reached 0.4–0.5, the cultures were diluted 10 times with LB and H 2 O 2 was added to the final concentrations of 0.1 mM. These bacterial cultures were incubated for 30 minutes at 37°C with shaking. Catalase was added to each tube to a final concentration of 4 μg/ml. Ten millilitres of treated and control cultures were collected, immediately centrifuged at 10 000 x g for 2 minutes and the supernatant was removed. The bacterial pellets were resuspended in 1 ml LB and mixed with 1 ml of RNAprotect Bacteria Reagent (Qiagen, Germany), incubated during 2 minutes and centrifuged at 10 000 x g for 2 minutes at room temperature. The supernatant was discarded and the bacterial pellet was immediately frozen and stored at -80°C until RNA extraction.

RNA was isolated using RNeasy kit (Qiagen, Germany) according to the manufacturer’s instructions and eluted in 50 μl of RNase-free water. The nucleic acid yield and purity were determined by measuring the optical density at A260/280 using Nanodrop spectrophotometer (Thermo Scientific). RNA samples were treated with TURBO DNase (Life Technologies, Germany). Briefly, 10 μg of RNA were used in a total volume of 500 μl containing 20 units of TURBO DNase, incubated for 30 minutes at 37°C, immediately followed by RNeasy (Qiagen, Germany) clean-up and elution in 30 μl of RNase-free water. Following DNase treatment, RNA integrity was assessed using Agilent RNA 6000 Nano kit and 2100 Bioanalyzer instrument (both Agilent Technologies, USA). All samples had RIN values above 8.

For cDNA synthesis, total RNA (250 ng per reaction) and random primers were used for cDNA synthesis using High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Applied Biosystems, Germany). Initially, to ensure linear conversion of the transcripts, the dynamic range of reverse transcription reaction was tested by performing a standard curve with cDNA, synthesised using various input amounts of pooled RNA. To obtain a sufficient amount of cDNA, several batches of 20 μl RT reactions were pooled, diluted 50-fold with RNase-free water and stored in single-use aliquots at -80°C until further use. All 21 samples were tested for presence of contaminating genomic DNA by running the mdoG assay with cDNA and the respective no reverse transcription (-RT) controls. There was no amplification in the majority of–RT controls. In–RT samples with detectable amplification, difference in Ct values when compared with +RT varied between the samples, but was no less than 10 cycles for all of them with the lowest Ct values in–RT control samples ≥ 30.

For primer design, Escherichia coli strain K-12 MG1655 complete genome (accession U00096) sequence was downloaded from NCBI database ( www.ncbi.nlm.nih.gov ) and used as reference. Target sequence accession number, and primer sequences for each assay can be found in S11 Table . Primers were designed using Primer Express software (Applied Biosystems, Germany) and optimised for annealing temperature of 60°C. Each primer pair and amplicon were checked for secondary structure formation using Oligo Tool (Integrated DNA technologies, USA, http://eu.idtdna.com/analyzer/Applications/OligoAnalyzer/ ).

Quantitative Real-time PCR reactions were prepared manually in a total volume of 10 μl by mixing 5 μl 2x KAPA SYBR FAST ABI Prism master mix (KAPA Biosystems, Germany), 0.2 μl forward and reverse primer mix (10 μM each primer), 2.8 μl RNase-free water and 2 μl cDNA in MicroAmp Fast Optical 48-wells reaction plates (Applied Biosystems, Germany). PCR reactions were set up in the Fast mode using StepOne thermocycler (Applied Biosystems, Germany) with the following cycling conditions: 95°C 3’, 40X (95°C 3”, 60°C 20”), melting curve analysis. Each assay was run in duplicate. No template controls (NTC) were included each time. Presence of a single specific product was verified by running a melt curve analysis followed by visualisation of the qPCR product in a 2% agarose gel stained with SYBR Safe DNA Gel stain (Life Technologies). Additionally, each PCR assay was tested for reaction efficiency as follows: equimolar amounts of cDNA from all 21 samples were pooled together and used for the preparation of the standard curve by serial dilution (1:3) over five dilution points and run in triplicate. Expression of target genes was normalised to the expression levels of three reference genes ( arcA , mdoG and tus ), selected based on the assessment of the expression stability across all experimental conditions using BestKeeper software [ 71 ]. Reaction efficiency information inferred from the standard curve data was used to correct for differences in amplification efficiencies in the REST 2009 software [ 72 ]. Default settings (2000 iterations) were used for randomisation and bootstrapping analysis to test significance of gene expression. Expression values with p -values ≤0.05 were assigned as differentially expressed. We followed the Minimum Information for Publication of Quantitative Real-Time PCR experiments (MIQE) guidelines-compliant check-list [ 73 ].

For data analysis, qPCR Amplification curves were first visually examined in the StepOne software (Applied Biosystems, Germany). No baseline and threshold line adjustments were necessary. Ct values of the technical replicates were averaged and used for relative gene expression analysis in the REST 2009 software (Qiagen, Germany) [ 72 ]. Expression of target genes was normalised to the expression levels of three reference genes ( arcA , mdoG and tus ), selected based on the assessment of the expression stability across all experimental conditions using BestKeeper software [ 71 ]. Reaction efficiency information inferred from the standard curve data was used to correct for differences in amplification efficiencies in the REST 2009 software. Default settings (2000 iterations) were used for randomisation and bootstrapping analysis to test significance of gene expression. Expression values with p -values ≤0.05 were assigned as differentially expressed. We followed the Minimum Information for Publication of Quantitative Real-Time PCR experiments (MIQE) guidelines-compliant check-list [ 73 ].

Transcriptome sequencing

The transcriptome sequencing was carried out on samples treated with 0.1 mM H 2 O 2 during the experiment of memory decline. The time point used corresponded to 120 minutes after removal of the treatment. Bacterial cells that were kept in low density (0.05 OD 600 ) were concentrated 10 times and the pellets were resuspended in 1 ml of lysis buffer. Both, small RNA fraction and the large one were isolated using the microRNA & small RNA Isolation kit (Thermo Scientific, Germany). Traces of genomic DNA were removed from 10 μg of RNA by digestion in a total volume of 500 μl containing 20 units of TURBO DNase, incubated for 30 minutes at 37°C, immediately followed by RNeasy (Qiagen, Germany) clean-up and elution in 30 μl of RNase-free water. Following DNase treatment, RNA integrity was assessed using Agilent RNA 6000 Nano kit and 2100 Bioanalyzer instrument (both Agilent Technologies, USA). Both fractions were depleted from ribosome RNA using the Ribo-Zero Depletion Kit for Gram-negative bacteria (Illumina, USA). Libraries were prepared using a TruSeq Stranded Total RNA library preparation kit (Illumina, USA) and were sequenced on a MiSeq platform. All sequences are available from the NCBI SRA under BioProject accession PRJNA485867.

Evolution experiment

Before the beginning of the experiment, five independent clones of E . coli MG1655 were pre-adapted to the experimental conditions such as culture medium, temperature and oxygen level. The pre-adaptation was carried out by diluting an overnight culture of each clone 1:1000 followed by incubation at 37°C in the plate reader with the same shaking conditions used during the rest of the experiment in 0.2 ml in fresh-made LB. The bacteria were cultured by serial passage every 24 hours using a bottleneck of 1% (1/100 dilution) to allow for approximately fifty generations during 10 days. Contamination checks by plating out on LB and cryopreservation of culture aliquots at -80°C in LB containing 15% glycerol solution were carried out periodically. All lines showed similar fitness and five independent colonies (one per line) were selected for the evolution experiment. The experiment was performed at 37°C with periodical shaking in a microplate reader (Synergy H1, Biotek, Germany). We used flat-bottom polystyrene 96-well plates which lids had anti-evaporation rings (Greiner Bio-One, Germany). A final volume of 200 μl per well was used and we founded 60 independent populations: 20 populations that we evolved under priming conditions, 20 populations under non-priming conditions and 20 non-evolving populations that were only serially passed. Growth curves were generated by taking measurements of OD 600 every 20 minutes (preceded by a brief shaking of 5 seconds). Every day, the experiment consisted of two stages. Every morning a V-bottom plate containing 190 μl of LB was inoculated with 2 μl of overnight culture and incubated for 2 hours to reach an OD 600 between 0.2 to 0.6. At this point, 3 μl of LB containing the corresponding priming concentration of H 2 O 2 (for populations to be primed) or only LB (control populations) were added to each well. The plate was then incubated for 30 minutes, centrifuged and the supernatant removed using a Costar 8-channel vacuum aspirator (purchased from Sigma Aldrich, Germany) equipped with disposable tips. The pellets were resuspended in 200 μl of fresh LB and 190 μl were transferred to a new flat-bottom plate. Cells were allowed to recover for 30 minutes at 37°C and challenged with a ten-fold higher concentration of the priming one. The dish was placed in the plate reader and the growth was followed as described above during 20 hours. The serial passage started at 50 μM of H 2 O 2 . Next day, the procedure was identical for priming and triggering concentrations that were doubled every 24 hours to reach a final challenging concentration (trigger) of 32 mM H 2 O 2 where all bacterial population went extinct. Before each passage, 20 μl per population were added to 180 μl of sterile 0.9% NaCl and then serially diluted, plated and inspected for contamination and population extinction.

DNA isolation

Genomic DNA samples for whole genome sequencing were isolated using in house method based on fast phenol:chloroform extraction, removal of RNA and ethanol precipitation. The DNA quantity and quality were estimated by measuring the optical density at A260/280 using a Nanodrop 2000 (Thermo Scientific, Germany) and agarose gel electrophoresis.

Genome re-sequencing

We sequenced the total genomic DNA from sixty populations from the evolution experiment, all from the final passage before extinction (20 evolved from the priming regime, 20 from the non-priming regime and 20 control strains). TruSeq DNA PCR-free libraries were constructed according to the manufacturer’s instructions and sequenced for 600 cycles using a MiSeq at the Berlin Center for Genomics in Biodiversity Research. Sequence data are available from the NCBI SRA under BioProject accession PRJNA485867. The haploid variant calling pipeline snippy [ 74 ] was used to identify mutations in the selection lines. Snippy uses bwa [ 75 ] to align reads to the reference genome and identifies variants in the resulting alignments using FreeBayes [ 76 ]. All variants were independently verified using a second computational pipeline, breseq [ 77 ].

Determination of H 2 O 2 -induced mutagenesis

This procedure was carried out following similar protocols to previous studies with some modifications [ 78 , 79 ]. Five independent cultures (5 ml each one) of E . coli MG1655 were grown in fresh LB medium to an OD 600 of ~0.2. Then, each culture was diluted 10 times (volume was increased to 50 ml) with LB and divided into two separate sets of 25 ml per culture each one. One set, consisting of five cultures of 25 ml was treated with 0.1 mM H 2 O 2 (primed) while the second set of culture remained untreated (naïve). After 30 minutes, both cultures were treated with 1 mM H 2 O 2 during another 30 minutes. Then, H 2 O 2 was removed, first, by quickly adding 4 μl/ml of catalase and, second, by collecting the cells by centrifugation and washing them with one ml of fresh LB. Another set of tubes received no treatment and were used to determine the basal frequency of mutants. For determination of H 2 O 2 -induced mutation frequency, a 4 ml tube of fresh LB was inoculated with 1 ml of washed bacteria from each culture. The cultures were allowed to recover overnight for 16 h at 37°C. Serial dilutions of each culture were plated next day onto LB plates containing 100 μg/ml of rifampicin or without antibiotic to estimate the viability. The three groups were compared among them using a Welch’s test.

Attachment assays

Attachment assays of an E . coli mutant in fimE and control strains to microscope glass slides were investigated. The experiment consisted of dipping sterile microscope slides into 50 ml Falcon tubes containing 10 ml of mid-exponential phase cultures of E . coli (0.5 OD 600 ), five culture for each group. The slides were incubated during 1 hour. In the last 10 minutes of the incubation, 10 μl of the LIVE/DEAD BacLight Bacterial Viability Kit (Thermo Scientific, Germany) was added to each culture following the instructions of the manufacturer. The slides were mounted with a glass coverslip and fluorescent images were taken of several fields with a simultaneous acquisition in red and green fluorescent channels with a Nikon Ti-2 inverted microscope (Nikon, Japan). Cells were observed with the 100× objective and controlled by Nis Element AR software.

Bacterial swimming assay

Swimming motility of mutants in the upstream region of fldhC and their complementation analysis were carried out on swimming plates containing swimming medium (0.5% bacto-tryptone and 0.3% agar, both from Sigma Aldrich, Germany). Briefly, 1 ml of overnight cultures per strain was centrifuged and resuspended in their supernatant adjusting their final OD 600 to 4. From these OD-adjusted cultures, 2 μl from each one were inoculated into the swimming plates in pairs and bacteria were allowed to grow and swim for 4 h at 37°C. For complementation we used the plasmid pVN15 (pBAD24 carrying flhDC operon genes) and the vector pBAD24 was used as control [ 80 ]. For contrast purposes, another 5 ml of swimming medium containing 0.25 mM isopropyl-β-D-thiogalactopyranoside (IPTG) and 4 μg/ml 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside (X-gal) were added carefully covering the entire surface of each plate. After the top layer solidification, the plates were incubated for 15 minutes at 37°C and 1 hour at 4°C before taking photographs. The combination of IPTG and X-gal turn the swimming surface into blue colour.

Statistical analysis

For point-to-point comparisons in sensitivity and priming killing curves a Welch’s test was used. Growth curve analysis was carried out using the Growthcurver package for R. For survival analysis we used a log-rank test. Some specific analyses are mentioned elsewhere in the manuscript. All tests were performed with software R [ 81 ] except mutational spectrum analysis that was implemented using the ad hoc software iMars [ 48 ].

Supporting information

Panel A represents a graphical model fitting of individual curves plotted by Growthcurver R package [ 60 ]. Panel B shows the average growth curves from 40 independent replicas per each situation (0.1 mM H 2 O 2 versus control).

Asterisks represent significant differences between the wild-type (wt) strain and its derivatives mutants (Welch’s test, one asterisk for p <0.05 and two asterisks for p <0.01).

The extinction was perceived by negative growth in the next passage and by the absence of growth in LB plates during contamination controls. Non-evolving population control (grey line, 20 populations) is presented. Evolvability differs between the two naïve population groups (red and magenta lines) and primed populations (blue line). Equal letter represents no statistical differences while the same letter indicates significant differences in pair-wise comparison (Log-rank test, p < 0.05). Differences with non-evolved populations were not determined.

Cells were collected 30 minutes after exposure. Error bars represent the standard error of the mean of three independent biological replicates, each biological replicate is the average of three technical repetitions.

Naïve and primed cells (pre-treated with 0.1 mM, 30 minutes in advanced) cultures challenged with 1 mM, allowed to recover and plated in rifampicin (100 μg/ml). The basal level of mutagenesis for non-pre-treated, non-challenged cells is also shown. Every sample consisted of five independent replications. Letters denote significant differences (Welch’s test, p = 0.03 for basal level versus primed, p <0.01 for both basal versus naïve and primed versus naïve).

The growth curve parameters were estimated with the Growthcurver R package [ 61 ]. Only carrying capacity and the areas under the curve have shown significant differences with a small effect.

H 2 O 2 concentrations were determined for 0, 15 and 30 minutes after the addition of H 2 O 2 using the Pierce Quantitative Peroxide kit (Thermo Scientific, Germany). The shown values represent the mean of the supernatant from three individual cultures and their standard deviations.

Bacteria were sampled 30, 60, 90, 120 and 150 minutes after removal of the treatment. Each treatment group consisted of six independent replicates and bacteria before treatment (T0) were used as control. Statistical analysis used student t-test and false discovery rate for correction of the p-values (data analysis using Maxquant and Perseus software for label-free quantification of proteins with LC-MS).

Each treatment group consisted of six independent replications and bacteria before treatment (T0) were used as control. Statistical analysis used student t-test and false discovery rate for correction of the p-values (data analysis using Maxquant and Perseus software for label-free quantification of proteins with LC-MS).

The predictions were carried out using the online tool ProtParam [ 69 ].

Acknowledgments

We would like to thank Heidrun Häweker and Elisa Bittermann for help with library preparations and technical assistance. We are grateful to Drs Gisela Storz (NIH, Bethesda, USA) and Karen Fahrner (Harvard University, Boston, USA) for kindly supplying the strains of E . coli OxyS deficient mutant and the plasmid carrying FlhDC operon respectively. For mass spectrometry (M.E. and C.W.) we would like to acknowledge the assistance of the Core Facility BioSupraMol supported by the Deutsche Forschungsgemeinschaft (DFG). We also thank to Francesca Zucchetti Villagarcía and Flor I. Arias-Sánchez (from Freie Universität Berlin) for valuable help with the proofreading of the article.

Funding Statement

ARR and JR were supported SFB973 from Deutsche Forschungsgemeinschaft ( http://www.sfb973.de/ ), project C5 ( http://www.sfb973.de/members_staff/c5/index.html ). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data Availability