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Our office will be closed for the summer holiday from 12th - 27th of July (both days included).

We wish everyone a lovely summer!

The Loligo^® team

The microplate respirometry system already have a long and well proven track record for providing high throughput oxygen consumption measurements  in a range of different small organisms (e.g., Daphnia, Zebrafish embryos, marine invertebrate larvae, etc.).

In this application note, we demonstrate how the microplate system can also be applied for high throughput respiration measurements of microorganisms in suspension, here in corrosion samples dominated by the bacterium Acidithiobacillus thiooxidans.

Sulfide oxidation by A. thiooxidans at low pH can lead to the production of sulfuric acid, which is very problematic for concrete corrosion. 

H₂S+ 2O₂ → H₂SO₄ ​ (sulfuric acid)

As the sulfide oxidation is driven by an O₂ reduction and consumption, this process can be investigated by following the change in O₂ over time with the use of the microplate respirometry system.

The aim in this setup and demonstration was to investigate the inhibitory effect of nitrite on this sulfide oxidation under low pH (~ 2) in samples of concrete corrosion materials, using the microplate respirometry system.

Approach

Corroded concrete material, softened and structurally deteriorated as they have been exposed to microbial induced corrosion (expected to be dominated by A. thiooxidans), was mixed with water, sulfuric acid, sulfide and with different concentrations of nitrite (NaNO₂). The samples were vortexed to mix and aerate the samples before being distributed across the glass microplate with 1,700 µL wells. To support continuous agitation, one glass bead was included in each well before the wells were filled with sample solution and sealed, and the plate and reader were placed on an orbital rotating shaker table.

After this, the change in the oxygen saturation in the 24 wells (with different nitrite concentrations) was monitored and logged using the MicroResp software. Below is an image of the results/output in the MicroResp software. The first column is the control wells (no nitrite added), and each of the subsequent columns are wells with increasing nitrite concentrations. The unit mg/L refers to mg NO₂^--N / L, which represents the concentration of the added nitrogen (N) in the form of nitrite (NO₂^-) in the samples.

These results clearly show that O₂ consumption decreased with increasing nitrite concentration. In control wells and wells with lower nitrite concentrations, most O₂ was consumed within one hour. For the highest nitrite concentrations, only limited O₂ was consumed within one hour. These results demonstrate an inhibitory effect of increasing nitrite concentration on microbial oxygen consumption, and thus, that this microbial activity could be evaluated using the microplate system.

Based on the MicroResp data output, a figure like this can be created:

Figure created based on the MicroResp analysis software output. Well D1
excluded due to a trapped air bubble reoxygenating the sample.

This data output could be used to create dose response curves, here based on the first 30 minutes of monitored oxygen consumption:

Inhibition of oxygen consumption compared to controls (without added nitrite) at increasing
nitrite concentration after the first 30 minutes. Values are average of the four replicates ± 1 SD.

The microplate system was also used to investigate microbial oxygen consumption over a longer time frame, here for about 6 hours:

These experiments were carried out as a part of the master’s thesis work by Sune Popp Hinke and Line Gade Frahm together with Associate Professor Asbjørn Haaning Nielsen at the Department of the Built Environment, Aalborg University. A big thanks to all for testing our system to monitor microbial oxygen consumption.

Further insights on the project and challenges associated with acid formation, by Professor Asbjørn Haaning Nielsen:

This application note demonstrates how a microplate-based respirometry system can be used for high-throughput measurements of microbial respiration in concrete samples undergoing biogenic sulfuric acid (BSA) attack. Such environments are typically colonized by the acidophilic, sulfur-oxidizing bacterium Acidithiobacillus thiooxidans, a key driver of BSA.

At the low pH characteristic of BSA-affected concrete surfaces, A. thiooxidans oxidizes hydrogen sulfide (H₂S) to sulfuric acid (H₂SO₄):

H₂S+2 O₂ → H₂SO₄ ​

The acid aggressively dissolves the calcium-rich cement matrix, forming gypsum and ettringite whose expansion leads to cracking and loss of structural integrity. The microplate system enables rapid, parallel monitoring of oxygen consumption, providing high-resolution insight into sulfur-oxidizing activity directly in BSA-affected concrete. In the present study, we examine how nitrite inhibits the BSA process by reducing the sulfur-oxidizing activity of A. thiooxidans and thereby slowing sulfuric acid generation.

If you too are interested in the microplate respirometry system for oxygen consumption rate measurements in suspensions of bacteria, algae, yeast, protozoa etc., please reach out to our product specialist Dr Louise Vinther Grøn (lvg@loligosystems.com), who will be happy to discuss your experimental needs and considerations.

We are looking forward to seeing even more studies on microbial oxygen consumption with the use of the MicroResp system in the future.

We are happy to welcome and introduce our new product specialist, Louise Vinther Grøn, to the Loligo Systems team. We have said goodbye to Andreas Mørck, who after 8 years in this position is moving to a new position in a biotech company. We thank him for all his great work here at Loligo Systems and wish him all the best of luck in his new job!

Louise joins our team from Aarhus University, where just last week she successfully defended her PhD in Biological and Chemical Engineering. During her PhD studies, Louise worked with acetogenic microorganisms in bioelectrochemical systems. From this work, she has a strong background in the integration of measurement and sensor technologies into specialized systems to investigate biological processes, and before that, an educational background in Biology. Building on this experience, Louise will now provide our customers at Loligo Systems with valuable, efficient and free support on our systems – as we have always offered.

With her background in microbiological systems, Louise will also explore how our measurement systems can be applied for microbial oxygen consumption and respirometry. If you are curious about the possibilities of using our systems for your microbiological experiments, please reach out to Louise!

In this article, we showcase how the microplate respirometry system provides an easy to use, high throughput respirometry tool for determining oxygen consumption rates in various life stages of terrestrial insects, here using fruit flies (Drosophila melanogaster and Drosophila littoralis). The project was performed at Aarhus University in Denmark in collaboration with Prof Jesper G. Sorensen at Aarhus University in Denmark.

The first part of the project looked at the respiration rates in eggs, third instar larvae, and pupae of D. melanogaster. The fruit flies were maintained at 19 °C, and were in the following life stages:

• melanogaster eggs: 0-12 hours old
• melanogaster larvae: 6-7 days since egg stage
• melanogaster pupae: 8-9 days since egg stage

The first trial had 10 x eggs + 10 x pupae (+ 4 x blanks). The eggs and pupae were transferred directly from their food solution/colony and placed individually in a well in the 80 µl glass microplate. The wells were then closed using gas-tight non-toxic sealing film, and placed onto the microplate reader. The microplate reader and glass plate were then covered with an opaque plastic box, to prevent ambient light from interfering with the optical oxygen sensor inside each well, and left at room temperature (21 °C). Here is a picture of the setup:

The MicroResp™ v1.1 software was used to calibrate the oxygen sensors, set up the plate and experiment settings, and to log data. Here are the exported data graphs:

Oxygen data graph for 10 x eggs (dark golden), 10 x pupae (purple), and 4 x blanks (black) measured over 2 hours and 18 min.

The next trial used 20 x third instar larvae (dark cyan) and 4 x blanks (black), and data was logged for 1 hour and 35 min.:

A data plot of the MO₂ values shows oxygen consumption rates in the pupae (second from right) and larvae (first from right) but no consumption was detected in the eggs and blanks (far left):

The next part of the project investigated the respiration rates in adult fruit flies, here D. littoralis. Besides determining the oxygen consumption rates of adult individuals, we also wanted to look at the effects on MO₂ from anesthetization with CO₂ – a common anesthetic used when handling live fruit flies. An individual adult fruit fly was transferred using an aspirator tube from their maintenance chamber at 21 °C and then directly into a well or anesthetized for 30 sec. with CO₂ prior to transfer. The 200 µl glass plate was then closed with sealing film and placed under a box.

The MicroResp™ v1.1 software was used to calibrate the oxygen sensors, set up the plate and experiment settings, and to log data. Here are the exported data graphs:

Oxygen data graph for 12 x adult flies (green), 8 x adult flies anesthetized with CO₂ (blue), and 4 x blanks (black) measured over 4 hours and 41 min. A data plot of the MO₂ values shows no apparent difference between non-anesthetized (mid) and anesthetized individuals (right):

In conclusion, the microplate respirometry system was able to dertemine oxygen consumption rates in various life stages of fruit flies in a relative short period of time.

Want to learn more about the microplate respirometry system?

• Microplate respirometry system product page
• Learn more about the MicroResp™ software
• User case – Testing microplate system against traditional gas analysis
• User case - Antarctic insect metabolism

We are excited to offer users of Loligo® software this new free and useful tool.

The LoliGO App is a smartphone application for Android and iOS that allows users to remotely monitor their experiments while having the option of getting push notifications in case of power outs in the lab, computer failures, or alarms based on water quality thresholds – like if the temperature gets critically high in your respirometry chambers.

The LoliGO App uses a secure cloud-based service to handle data sent from the Loligo® software and to the App. Users can set up a user profile, and multiple users can access the same user profile at the same time (if they know the password!) aka you can have a shared lab profile.

You cannot alter or stop experiments from the app, and you cannot export or otherwise gain access to the raw data from the app. Completed experiment data will be automatically deleted from the app after 14 days.

The LoliGO App is free to use, and is currently compatible with AutoResp™ v3, and the app will be available with future updates of other Loligo® software soon.

Download the latest version of the LoliGO App:

• LoliGO for Android (on Google Play)
  
  
• LoliGO for iOS (on App Store)

Once again, we are honored to be supporting the scientific community. In 2024, we are sponsoring a list of symposia, workshops, and meetings, and we encourage all interested to take a closer look at the following events:

• We have sponsored travel money for invited speakers at the very interesting SEB 2024 Symposium ”Balancing energy acquisition, expenditure, and allocation in an ever-changing world” through Dr. Amanda Pettersen (co-chairs: Neal Dawson, Ed Ivimey-Cook), and we hope to see you all for a great event in Prague, July 2-5!
  
  
• We have donated cash to support students going to the CSZ-SCZ annual meeting on May 6-10, 2024, in Moncton, NB.
  
  CSZ-SCZ 2024 - Home / Accueil (cszsczmoncton2024.com)
  
  
• Once again, we have accepted to be ICBF 2024 Student Travel Sponsors to help young people participate in this biennial meeting.
  
  International Congress on the Biology of Fish 2024 | 15th (michiganseagrant.org)
  
  We also support this meeting through a Lake Superior Sponsorship and participate as exhibitors as we did for nine (9) consecutive ICBF meetings
  
  See you in Ann Arbor Jun 23-27!
  
  
• We are proud sponsors of the 47th annual Larval Fish Conference in Huron, Ohio, on May 12-16, and hope that it will be a great event.
  
  47th Larval Fish Conference
  
  
• We are also proud sponsors of the upcoming Midwest Zebrafish Conference 2024 taking place June 7-9 at Washington University in St. Louis.
  
  Check out the meeting right here: Midwest Zebrafish Meeting 2024
  
  
• We give financial support to a workshop on metabolism in animal ecophysiology arranged by Dr. Christel LeFrancois, Dr. Marie Vagner (Brest) and Dr. Loïc Teulier (Lyon). This seminar will take place on October 2-3, 2024, in La Rochelle, France.
  
  For more details, please visit: https://www.rt-metabolica.cnrs.fr/

Image: Male toadfish showing strong paternal care for 10 DPF brood. Image by Martin Grosell, RSMAS, University of Miami.

If you are looking for a solid fish respirometry and hypoxia paper with thorough explanations on methods, protocols, and data analysis, look no further than this 2023 publication by LeeAnn Frank, Joseph Serafy, and Prof. Martin Grosell from the Rosenstiel School of Marine, Atmospheric, and Earth Science, University of Miami.

A large aerobic scope and complex regulatory abilities confer hypoxia tolerance in larval toadfish, Opsanus beta, across a wide thermal range

Frank, LeeAnn; Serafy, Joseph; Grosell, Martin (2023)

Science of The Total Environment

Link to article

The team of researchers provides an easy-to-follow methodical description on how to measure standard metabolic rate (SMR), routine metabolic rate (RMR), maximum metabolic rate (MMR), and aerobic scope (absolute and factorial) as well as how to assess the regulation index, P₅₀, and Excessive Post Hypoxia Consumption (EPHOC) data in larval toadfish (Opsanus beta) over a range of their environmental temperatures. In addition to the detailed materials and method section, the authors supply the article with a manageable overview on how to analyze such respirometry data and which data parameters to include. The data are effectively illustrated using an assortment of data graphs, plots, and tables.

If you have any questions about the paper, you are welcome to contact LeeAnn Frank at leeann.frank@earth.miami.edu.

Want to know more about the respirometry system used in this paper?

• Glass chamber respirometry systems
• Read more about the intermittent respirometry software, AutoResp™ v3
• Learn how to calculate the respirometry parameters in AutoResp™ v3
• More published papers on aquatic respirometry

In a peer-reviewed publication by Earls et al. (2023), a team of researchers from North Dakota State University and USDA compares the Loligo® microplate respirometry system with a traditional gas analyzer setup in a study measuring the metabolism of using alfalfa leafcutting bees (Megachile rotundata) and effects of temperature. The paper can be found here:

Effects of temperature on metabolic rate during metamorphosis in the alfalfa leafcutting bee | Biology Open | The Company of Biologists

Earls, Kayla N; Campbell, Jacob B; Rinehart, Joseph P; Greenlee, Kendra J

• Biol Open (2023) 12 (12): bio060213.
• https://doi.org/10.1242/bio.060213

Daisy-chained microplate reader with glass plate on top.

The Loligo® microplate respirometry system used in this study featured two daisy-chained microplate readers using glass plates with 940 µl wells for simultaneous measurements of up to 48 bees at a time. Both readers were controlled using our MicroResp™ software, which was also used for calculating the V̇O₂ slope and to randomly assign bees to the different wells.

A mix of brood cell and pupae M. rotundata in 940 µl wells. Photo by Kayla Earls.

The 48-chamber microplate setup was compared to a traditional closed respirometry setup with manual injections of air samples into a CO₂ analyzer (Li-Cor-7000, LI-COR Biosciences, Lincoln, NE, USA) and O₂ (Oxzilla FC-2 Differential Oxygen Analyzer, Sable Systems International, Las Vegas, NV, USA) gas analyzer.

The scientists found no significant differences between the V̇O₂ data from the two different setups when comparing mass-specific metabolic rate calculations:

Copied from Fig. 1. in the publication.

But the setup comparison shows a significant difference in the time (and resources) spent on collecting the data:

“Pupal bees in the traditional closed respirometry system remained in the [individual] syringes for longer periods (∼9 h) than bees tested in the microplate system (∼3 h) due to the differences in chamber size (3 ml syringes versus 940 µl wells) and detection limits of the oxygen analyzers.”

It is also worth mentioning that a microplate system can analyze up to 240 organisms at a time, while the bees in the traditional closed respirometry had to be analyzed one at a time. In other words, the time spent on collecting data is substantially shorter in the microplate system.

Most impressively, the researchers present a vast dataset on oxygen consumption rates in the leafcutting bees across a large range of temperatures (6 – 48 °C) and developmental stages and between sexes:

Copied from Fig. 2. in the publication.

Copied from Fig. 4. in the publication.

If you are interested in learning more about this project or its team, you are welcome to contact Kayla N. Earls (Kayla.Earls@wsu.edu).

Want to learn more about the microplate respirometry system?

• Microplate respirometry system product page
• Learn more about the MicroResp™ software
• Another microplate respirometry usercase featuring Antarctic insects