The authors thank Prof SV Albers (University of Freiburg), Prof Eveline Peeters (Vrije Universiteit Brussel), and Dr Rani Baes (Vrije Universiteit Brussel) for advice and the S.&#160;acidocaldarius DSM639 strain. This work was funded by a Royal Society Research Grant (awarded to DRG: RGS\R1\231308), a UKRI-NERC &#34;Exploring the Frontiers&#34; Research Grant (awarded to DRG and CGK: NE/X012662/1), and a Kuwait University PhD scholarship (awarded to ZA).

Experimental Evolution Protocol for Thermophile Adaptation Using Low-Cost Thermomixers

<ol>
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The authors do not declare any conflicts of interest.

This work has developed an experimental evolution protocol for thermophiles, here tailored for the archaeon S. acidocaldarius, but adaptable to other microbes with high-temperature growth requirements. This protocol builds on techniques initially designed for mesophilic bacteria but is specifically modified to overcome the technical challenges associated with high-temperature aerobic growth2,4,5,24. The creation of this method will enable the exploration of previously uncharacterized evolutionary dynamics that occur under extreme temperature conditions, resembling the conditions that potentially existed during the earliest stages of the evolution of life1.
There are several critical aspects of the protocol that must be observed to achieve reliable results from evolution experiments. The first is independence between lineages, which is achieved by streaking out to single colonies before initiating the experiment. This is imperative to ensure that any common mutations found in separate lineages have independent origins. If, instead, a bulk liquid culture is aliquoted to initiate the lineages, common genetic variants may be distributed to each population. A second key consideration is to control for contamination, which is achieved through maintaining a negative control free from culture and monitoring for growth. Unlike experiments with mesophiles, extremophile evolution experiments are unlikely to be contaminated from external sources, but cross-contamination between lineages may still occur. Maintaining a balance between aeration and evaporation is a further key consideration. S.acidocaldarius is an obligate aerobe and so requires oxygen for growth10,23. Manually piercing polypropylene microcentrifuge tubes and sealing them with gas permeable membrane21,25, was key to maintaining aeration without excessive evaporation due. This reduction can be likely attributed to the preservation of a uniform environment within the sealed tubes (data not shown).
Two aspects of the method may require adjusting or troubleshooting according to the specific environmental conditions used for the evolution experiment: the dilution factor and the time between transfers. In this method, a 1:100 dilution was performed every transfer, necessitating an average of log2(100) = 6.64 doublings to return to the same population size. This provides a good balance between the potential for adaptation (an optimal dilution factor is calculated by Wahl and Gerrish26) and experimental tractability. In principle, a strong dilution factor would enable more generations to occur per day, as it requires more doublings to return to the same population density (e.g., 1:1000 requires log2(1000) = 9.97 doublings on average). However, if fewer doublings are achieved than are required between transfers, the populations will gradually be washed out through dilution unless a beneficial mutation that decreases doubling time occurs. with populations transferred every 48 h, it is required that the doubling times be less than 8 h, which aligns with the estimated doubling time and previously published values for heterotrophic growth in S. acidocaldarius at 75 &#176;C (4-6 h)23,27. However, the gradual decrease in OD600nm over time for the constant 65 &#176;C and temperature drop treatments suggests that this may not be a long enough time to achieve the required number of doublings and that a longer transfer period would be beneficial.
Several important advantages of using thermomixers over traditional shaking incubators for experimental evolution are noted, particularly when investigating adaptation to temperature. Thermomixers present a cost-effective alternative to shaking incubators, with the added advantage of a reduced spatial footprint. These characteristics enable the efficient assessment of multiple temperature treatments in parallel. Thermomixers also exhibit lower energy consumption than shaking incubators, so their use can potentially reduce environmental impact when a modest number of tubes is incubated (e.g., fewer than 100). This disparity in energy consumption is particularly evident at temperatures relevant to thermophiles but is also applicable across a spectrum of more moderate temperature ranges (Figure 6), meaning thermomixers could also be adopted for reducing the environmental impact of mesophile experimental evolution. Collectively, these attributes suggest that using thermomixers in experimental evolution has the potential to improve the accessibility of this research methodology, particularly in resource-constrained environments, such as laboratories in developing countries and in primary and secondary education.
Despite providing a way forward for thermophile experimental evolution, the current method has some limitations relative to mesophile experimental evolution. Firstly, the number of replicate independent lineages that can be established using thermomixers is small relative to high-throughput methods involving microtiter plates, which are in widespread use for mesophiles4. Evaporation at high temperatures currently precludes the use of microtiter plates over the time window required to reach exponential growth. Secondly, estimating key growth curve parameters, such as growth rate, doubling time, and lag time, is made challenging as plate-based spectrophotometers that can operate at high temperatures (&#62;45 &#176;C) are not currently available for purchase. With the method outlined here, growth curve characterization at present requires labor-intensive destructive sampling and hence limits throughput. Finally, high-throughput techniques for assessing the competitive fitness of adapted isolates are not yet thoroughly developed for thermophiles. Particularly, the development of a thermostable fluorescent protein that can be used to differentiate between strains would enable high-throughput flow cytometry-based fitness assays28,29; recent developments of thermostable fluorescent proteins with different spectra raise this as a potential future development30,31,32. Increases in throughput are a logical next step for the further development of experimental evolution techniques for thermophiles, as it will enable evolutionary processes to be more completely described.

Early life on Earth may have originated in extreme environments, such as hydrothermal vents, which are characterized by extremely high temperatures and acidity1. Microbes continue to inhabit extreme environments, including hot springs and volcanic solfatara. Characterizing the evolutionary dynamics that occur under these extreme conditions may shed light on the specialized physiological processes that enable survival under these conditions. This may have wide-ranging implications, from our understanding of the origins of biological diversity to the development of novel high-temperature enzymes with biotechnological applications.
The understanding of microbial evolutionary dynamics in extreme environments remains limited despite its critical importance. In contrast, a significant body of knowledge about evolution in mesophilic environments has been acquired through the application of a technique known as experimental evolution. Experimental evolution involves observing evolutionary change under laboratory conditions2,3,4,5. Often, this involves a defined change environment (e.g., temperature, salinity, introduction of a toxin or a competitor organism)7,8,9. When combined with whole-genome sequencing, experimental evolution has enabled us to test key aspects of evolutionary processes, including parallelism, repeatability, and the genomic basis for adaptation. However, to date, the bulk of experimental evolution has been performed with mesophilic microbes (including bacteria, fungi, and viruses2,3,4,5, but largely excluding archaea). A method for experimental evolution applicable to thermophilic microbes would enable us to better understand how they evolve and contribute to a more comprehensive understanding of evolution. This has potentially wide-ranging implications, from deciphering the origins of thermophilic life on Earth to biotechnological applications involving &#39;extremozymes&#39; used in high-temperature bioprocesses10 and astrobiological research11.
The archaeon Sulfolobus acidocaldarius is an ideal candidate as a model organism for developing experimental evolution techniques for thermophiles. S. acidocaldarius reproduces aerobically, with an optimal growth temperature at 75 &#176;C (range 55 &#176;C to 85 &#176;C) and high acidity (pH 2-3)4,6,12,13,14. Remarkably, despite its extreme growth conditions, S. acidocaldarius maintains population densities and mutation rates comparable to mesophiles7,15,16,17,18. In addition, it possesses a relatively small, well-annotated genome (strain DSM639: 2.2 Mb, 36.7% GC, 2,347 genes)12; S. acidocaldarius also benefits from robust genome engineering tools, allowing for a direct assessment of the evolutionary process through targeted gene knockouts19. A notable example of this is the availability of genetically modified strains of S. acidocaldarius, such as the uracil auxotrophic strains of MW00119 and SK-120, which can serve as selectable markers.
There are significant challenges with conducting experimental evolution with thermophiles like S. acidocaldarius. Extended incubation at high temperatures required for these studies imposes considerable evaporation for both liquid and solid culturing techniques. Extended operation at high temperatures can also damage the traditional shaking incubators that are commonly used in experimental evolution in liquid media. Exploring multiple temperatures necessitates a substantial financial investment for acquiring and maintaining several incubators. Furthermore, the high energy consumption required raises significant environmental and financial concerns.
This work introduces a method to address the challenges encountered in performing experimental evolution with thermophiles like S. acidocaldarius. Building upon a technique developed by Baes et al. for investigating heat shock response14,21, the method developed here utilizes bench-top thermomixers for consistent and reliable high-temperature incubation. Its scalability allows for the simultaneous assessment of multiple temperature treatments, with reduced costs in acquiring additional incubation equipment. This enhances experimental efficiency, enabling robust statistical analysis and extensive investigation of factors influencing evolutionary dynamics in thermophiles22. Moreover, this approach significantly reduces the financial initial investment and energy consumption compared to traditional incubators, offering a more sustainable and environmentally friendly alternative.
Our method lays the groundwork for experimentally investigating evolutionary dynamics in environments characterized by extreme temperatures, which may have played a key role during the early stages of the diversification of life on Earth. Thermophilic organisms have unique properties, but their extreme growth conditions and specialized requirements have often limited their accessibility as a model system. Overcoming these barriers not only expands research opportunities for investigating evolutionary dynamics but also enhances the broader utility of thermophiles as model systems in scientific research.

<table><tbody><tr><td>0.22 &amp;mu;m syringe-driven membrane filters</td><td>StarLab</td><td>E4780-1226</td><td>For filter sterilising media components that cannot be autoclaved.</td></tr><tr><td>1 &amp;mu;L inoculation loops</td><td>Greiner</td><td>731161, 731165, or 731101</td><td>For inoculating cultures. Other loops can be used.</td></tr><tr><td>1000 &amp;mu;L pipette tips</td><td>StarLab</td><td>S1111-6811</td><td>Other pipette tips can be used.</td></tr><tr><td>2 mL microcentrifuge tubes</td><td>StarLab</td><td>S1620-2700</td><td>For culturing &lt;em&gt;S. acidocaldarius&lt;/em&gt; in thermomixers.</td></tr><tr><td>200 &amp;mu;L pipette tips</td><td>StarLab</td><td>S1111-0816</td><td>Other pipette tips can be used.</td></tr><tr><td>50 mL polystyrene tubes with conical bottom</td><td>Corning</td><td>430828 or 430829</td><td>Other tubes may be used. Check performance at 75 &amp;deg;C. Tubes with plug seal caps may not allow sufficient aeration; check before using.&amp;nbsp;</td></tr><tr><td>50 mL syringe</td><td>BD plastipak</td><td>300865</td><td>For use with syringe-driven filters.</td></tr><tr><td>96 well microtitre plates (non-treated, flat bottom)</td><td>Nunc</td><td>260860</td><td>For measuring OD at 600 nm in spectrophotometer.</td></tr><tr><td>Adjustable width multichannel pipette</td><td>Pipet-Lite</td><td>LA8-300XLS</td><td>Optional, but saves time when transferring between microcentrifuge and 96 well plates.</td></tr><tr><td>Ammonium sulfate ((NH&lt;sub&gt;4&lt;/sub&gt;)&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Millipore</td><td>168355</td><td>For Brock stock solution I.</td></tr><tr><td>Autoclave</td><td>Priorclave</td><td>B60-SMART or SV100-BASE</td><td>Other autoclaves can also be used.</td></tr><tr><td>Breathe-EASY gas permeable sealing membrane</td><td>Sigma-Aldrich</td><td>Z763624-100EA</td><td>Cut to size to use on pierced microcentrifuge tubes. If substituting other gas permeable memrbanes, ensure performance is adequate at 75 &amp;deg;C</td></tr><tr><td>Calcium chloride dihydrate (CaCl&lt;sub&gt;2&lt;/sub&gt;&amp;middot;2H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Sigma-Aldrich</td><td>C3306</td><td>For Brock stock solution I.</td></tr><tr><td>CELLSTAR Six well plates (suspension/non-treated)</td><td>Greiner</td><td>M9062</td><td>Other manufacturers&#039; six well plates can likely be substituted. Check performance at high temperatures.</td></tr><tr><td>Cobalt(II) sulfate heptahydrate (CoSO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;7H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Supelco</td><td>1025560100</td><td>For Trace element stock solution.</td></tr><tr><td>Copper(II) chloride dihydrate (CuCl&lt;sub&gt;2&lt;/sub&gt;&amp;middot;2H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Sigma-Aldrich</td><td>307483</td><td>For Trace element stock solution.</td></tr><tr><td>D-(+)-glucose anhydrous (C&lt;sub&gt;6&lt;/sub&gt;H&lt;sub&gt;12&lt;/sub&gt;O&lt;sub&gt;6&lt;/sub&gt;)</td><td>Thermo Scientific Chemicals</td><td>11462858</td><td>Other pentose and hexose sugars may also be used (e.g. D-xylose, D-arabinose). Glucose is not a preferred carbon source for &lt;em&gt;S. acidocaldarius&lt;/em&gt; (SV Albers, personal communication)</td></tr><tr><td>Double-distilled water (ddH&lt;sub&gt;2&lt;/sub&gt;O)</td><td></td><td></td><td></td></tr><tr><td>Gelrite</td><td>Duchefa Biochemie</td><td>G1101.1000</td><td>Gelrite (gellan gum) is used in place of agar to make solid media due to its higher melting point.</td></tr><tr><td>Glass 100 mm Petri dishes</td><td>Brand</td><td>BR455742</td><td>Glass Petri dishes are used because most standard polystyrene 90 mm Petri dishes deform at 75 &amp;deg;C (brand-dependent). Alternatively, six well plates can be used as these do not deform at high temperatures.</td></tr><tr><td>Incubator</td><td>New Brunswick</td><td>Innnova 42R</td><td>Other incubators can also be used. Check the operating temperature for equipment prior to purchase/use, as many incubators are not capable of temperatures higher than 65&amp;deg;C.</td></tr><tr><td>Iron(III) chloride hexahydrate (FeCl&lt;sub&gt;3&lt;/sub&gt;&amp;middot;6H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Supelco</td><td>103943</td><td>For Fe Stock Solution</td></tr><tr><td>Magnesium sulfate heptahydrate (Epsom salt) (MgSO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;7H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Sigma-Aldrich</td><td>230391</td><td>For Brock stock solution I.</td></tr><tr><td>Manganese(II) chloride tetrahydrate (MnCl&lt;sub&gt;2&lt;/sub&gt;&amp;middot;4H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Sigma-Aldrich</td><td>SIALM5005-100G</td><td>For Trace element stock solution.</td></tr><tr><td>Mini Smart Wi-Fi Socket, Energy Monitoring</td><td>Tapo</td><td>Tapo P110</td><td>To monitor energy consumtion&amp;nbsp;</td></tr><tr><td>N-Z-Amine A - Casein enzymatic hydrolysate&amp;nbsp;</td><td>Sigma-Aldrich</td><td>C0626-500G</td><td>N-Z-Amine-A is used as a source of amino acids.</td></tr><tr><td>Paper clip (or other sturdy wire)</td><td>none</td><td>none</td><td>For piercing 2 mL microcentrifuge tubes.</td></tr><tr><td>Potassium dihydrogen phosphate (Monopotassium phosphate) (KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>P0662</td><td>For Brock stock solution I.</td></tr><tr><td>Promega Wizard Genomic DNA Purification Kit</td><td>Promega</td><td>A1120</td><td>Optional, to extract genomic DNA in the lab</td></tr><tr><td>Sodium molybdate dihydrate (Na&lt;sub&gt;2&lt;/sub&gt;MoO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;2H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Sigma-Aldrich</td><td>M1651-100G</td><td>For Trace element stock solution.</td></tr><tr><td>Sodium tetraborate decahydrate (Borax) (Na&lt;sub&gt;2&lt;/sub&gt;B&lt;sub&gt;4&lt;/sub&gt;O&lt;sub&gt;7&lt;/sub&gt;&amp;middot;10H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Sigma-Aldrich</td><td>S9640</td><td>For Trace element stock solution.</td></tr><tr><td>Spectrophotometer</td><td>BMG</td><td>SPECTROstar OMEGA</td><td>For measuring OD at 600 nm. Other spectrophotometers that can read OD at 600 nm can be used.</td></tr><tr><td>Sulfuric acid (Diluted in a 1:1 ratio with water) (H&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Thermo Scientific Chemicals</td><td>11337588</td><td>Used to adjust pH of Brock stock solution II/III to a final pH of 2&amp;ndash;3.</td></tr><tr><td>Thermomixer</td><td>DLab</td><td>HM100-Pro</td><td>Other thermomixers can also be used; key consideration is the ability to maintain 65&amp;ndash;75 &amp;deg;C temperatures and 400 RPM</td></tr><tr><td>Uracil (C&lt;sub&gt;4&lt;/sub&gt;H&lt;sub&gt;4&lt;/sub&gt;N&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>U0750</td><td>Deletion of pyrE is a common genetic marker used in &lt;em&gt;S. acidocaldarius&lt;/em&gt;. Deletion strains must be supplemented with uracil for growth. Supplementation is not strictly required for the DSM639 wild-type strain, but is included here as future experiments may involve deletion strains.</td></tr><tr><td>Vanadyl sulfate dihydrate (VOSO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;2H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Sigma-Aldrich</td><td>204862</td><td>For Trace element stock solution.</td></tr><tr><td>Zinc sulfate heptahydrate (ZnSO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;7H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Sigma-Aldrich</td><td>221376</td><td>For Trace element stock solution.</td></tr></tbody></table>

adaptation at the extremes of life: experimental evolution with the extremophile archaeon sulfolobus acidocaldarius

The archaeon Sulfolobus acidocaldarius has emerged as a promising thermophilic model system. Investigating how thermophiles adapt to changing temperatures is a key requirement, not only for understanding fundamental evolutionary processes but also for developing S. acidocaldarius as a chassis for bioengineering. One major obstacle to conducting experimental evolution with thermophiles is the expense of equipment maintenance and energy usage of traditional incubators for high-temperature growth. To address this challenge, a comprehensive experimental protocol for conducting experimental evolution in S. acidocaldarius is presented, utilizing low-cost and energy-efficient bench-top thermomixers. The protocol involves a batch culture technique with relatively small volumes (1.5 mL), enabling tracking of adaptation in multiple independent lineages. This method is easily scalable through the use of additional thermomixers. Such an approach increases the accessibility of S. acidocaldarius as a model system by reducing both initial investment and ongoing costs associated with experimental investigations. Moreover, the technique is transferable to other microbial systems for exploring adaptation to diverse environmental conditions.

Growth curve measurements 
Growth curves for S. acidocaldarius DSM639 are shown in Figure 3A. Growth was found to be similar when comparing incubation using thermomixers with that in conventional incubators. Average growth rate parameters were estimated by fitting a logistic curve to each replicated growth curve and calculating the mean and standard error. Times to mid-exponential phase on the thermomixer and incubator were 27.2 h &#177; 1.1 h and 31.1 h &#177; 1.9 h, respectively. Estimated initial doubling times for the thermomixer and incubator at 75 &#176;C were 4.29 h &#177; 0.28 h and 4.19 h &#177; 0.44 h, respectively, which is consistent with previously published values24. The relationship between log10(OD600nm) and log10(CFU) was well characterized by a linear model (adjusted R2 = 0.82, F(1,22) = 104, p &#60; 0.00001, slope = 1.73 &#177; 0.17, intercept = 9.73 &#177; 0.14). The relationship between OD600nm and CFU is thus given by the formula CFU = 10(1.73 &#215; log10(OD600nm) + 9.73). An OD600nm of 0.3 thus corresponds to approximately 6.7 &#215; 108 CFU/mL (Figure 3B).
Temperature evolution experiment 
Three temperature conditions, constant 75 &#176;C, constant 65 &#176;C, and temperature drop (75-65 &#176;C, decreasing by 1 &#176;C every two transfers), were initiated using seven independent lineages derived from S. acidocaldarius DSM639. OD600nm measurements were taken following each transfer over the 45 days of the experiment (approximately 150 generations at 75 &#176;C) and are shown in Figure 4. OD600nm measurements taken across days are inherently noisy, as they may be subject to subtle differences in, for example, growth period, temperature, etc. However, measurements taken across days can still be useful to assess population viability, as well as give an indication as to whether fitness is improving over time. Lineages from the constant 75 &#176;C condition increased in OD600nm from an initial range of 0.125-0.3 to a range of 0.248-0.471 by the end of the experiment. This suggests that fitness has improved with this treatment. In contrast, lineages from the constant 65 &#176;C treatment displayed a drop in OD600nm, from an initial range of 0.018-0.087 to 0.008-0.04 at the final time point. This suggests that populations were not able to adapt to the constant 65 &#176;C temperature, although the fact that viable organisms could be recovered shows that the populations were not washed out through successive dilutions, suggesting some degree of adaptation. Finally, populations in the temperature drop treatment increased from the initial OD600nm range of 0.099-0.279 to 0.3-0.39 at Tx6 (corresponding to 288 h and 73 &#176;C in this treatment), followed by a steady decrease to a range of 0.003-0.024 at the final time point.
Growth/fitness assays 
Fitness assays were performed for each descendent&#160;population following the evolution experiments. OD600nm was assayed after 48 h growth for all seven independent lineages, followed by fitting linear models in R for each assay temperature, with &#39;selection environment&#39; as a main effect and &#39;replicate/thermomixer&#39; as a block effect. Growth of the ancestral strain was used as the reference level for treatment contrasts. The data are shown in Figure 5.
When assayed at 75 &#176;C, there were on average significant increases in fitness over the ancestral strain for lineages from the constant 75 &#176;C (t-test: t210=3.64, p=0.0003) and constant 65 &#176;C (t-test: t210=2.8, p=0.005) treatments, but not from the temperature drop treatment (t-test: t210=&#8722;0.87, p=0.38). When assayed at 65 &#176;C, on average, lineages from all treatments displayed an increase in fitness (constant 75 &#176;C lineages; t-test: t210=4.68, p&#60;0.0001, constant 65 &#176;C lineages; t-test: t210,=4.24, p&#60;0.0001, temperature drop lineages; t-test: t210=3.15, p=0.002). However, for both assay temperatures, there were considerable differences between lineages in their fitness (Figure 5). Some lineages did not differ considerably from the ancestral strain or had decreased in fitness; this was particularly evident for lineages from the temperature drop treatment.
It is worth noting that the relationship between OD600nm and CFU/mL might have changed during the evolution experiment. This can be assessed by determining growth parameters for evolved lineages (following steps 3.1-3.10).
Whole genome sequencing results 
Whole genome analysis was carried out using breseq (version 0.38.1)25 on the seven lineages from the constant 75 &#176;C condition using the reference genome of S. acidocaldarius DSM639 (RefSeq accession NC_007181.1). A variety of mutations were revealed involving insertions, deletions, and single nucleotide polymorphism (SNP) across the genomes of all the descendent&#160;lineages (Table 2). Multiple insertion and nonsynonymous mutations were in protein-coding genes, as well as intergenic regions which may influence gene expression due to frameshifts in genes&#39; promoter regions. Some of the mutations were consistent across multiple descendent&#160;lineages in genes involved in various functions like cell wall biosynthesis, transcription, metabolism, cellular transport, and catalytic activity (Table 2). Among these mutations was a large deletion of 54,667 base pairs in five out of the seven lineages; this was confirmed by a missing coverage evidence plot for each population (frequency ranging from 93.2% to 100%). The deleted region equates to a loss of 53 genes; the roles of these genes in adaptation will be investigated in future studies. Some differences were noted between the used isolate of DSM639 and the published reference sequence (shown in Supplementary Table 1).
Energy consumption of shaking incubator versus thermomixer 
The energy consumption of a shaking incubator was compared to a thermomixer using a commercially available energy monitoring smart plug across a range of common incubation temperatures and the 75 &#176;C temperature used here. At 75 &#176;C, the thermomixer consumed approximately 1/40th the energy of a traditional shaking incubator (Figure 6), suggesting thermomixers as a potential means of reducing the carbon footprint associated with experimental evolution.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66271/66271fig01v2.jpg" /> 
Figure 1: Flow chart illustrating the evolution experiment protocol across three temperature treatments. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66271/66271fig01largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66271/66271fig02v2.jpg" /> 
Figure 2: Flow chart illustrating the steps of the growth/fitness assay protocol. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66271/66271fig02largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66271/66271fig03.jpg" /> 
Figure 3: Determination of key growth parameters and comparison of incubation devices for S. acidocaldarius DSM639. Three replicate cultures were grown at 75 &#176;C in 7 separate tubes. Destructive sampling was used to measure (A) OD600nm (means &#177; standard error of n=3 technical replicates; some error bars are smaller than plotting symbols); curves represent fitted logistic growth models and (B) colony forming units (CFUs); lines represent fitted log-log linear regression models). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66271/66271fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66271/66271fig04.jpg" /> 
Figure 4: Representative results for optical densities (OD600nm) obtained during evolution experiments. Optical densities of independent lineages measured during the evolution experiments under three temperature treatments (constant 65 &#176;C, constant 75 &#176;C, drop 75 &#176;C-65 &#176;C) conducted over approximately 150 generations. Curves depict Loess smooths over time for each independent lineage. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66271/66271fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/66271/66271fig05.jpg" /> 
Figure 5: Representative results for growth assays. Growth assays for independent lineages derived from S. acidocaldarius DSM639 following temperature evolution experiments (constant 65 &#176;C, constant 75 &#176;C, drop 75 &#176;C-65 &#176;C) in comparison to the ancestor strain. For all lineages, growth was assayed at 65 &#176;C and 75 &#176;C. Colored points show the mean &#177; standard error of technical replicates (shown in grey, n = 12 for the ancestor and n = 3 for each evolved lineage). The grey bar denotes the mean &#177; standard error of the ancestral fitness. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66271/66271fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/66271/66271fig06.jpg" /> 
Figure 6: Energy consumption of traditional incubator versus thermomixer devices. Energy consumption was recorded using a commercially available energy-monitoring smart plug over 2 h. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66271/66271fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Media recipes and stock solutions required to grow S. acidocaldarius. All media and stock solutions should be made with double-distilled H2O (ddH2O) and then sterilized either by autoclaving or filter sterilizing through a 0.22 &#181;m filter, as indicated. Please refer to section 1 of the protocol for a detailed description of how to prepare all media and stock solutions. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66271/Table 1.xlsx" target="_blank">Please click here to download this Table.</a>
Table 2: Representative results for whole genome sequencing of descendent&#160;lineages. Mutations found in descendent&#160;lineages descendant from S. acidocaldarius DSM639 from constant 75 &#176;C treatment. Mutations indicate changes relative to the direct ancestor of the lineage, which possesses several changes relative to the reference sequence for S. acidocaldarius DSM639 (RefSeq NC_007181; mutations in the ancestor are shown in Supplementary Table 1). n indicates the number of lineages in which a mutation was found. &#8594; gene on the &#39;forward reading frame&#39;; &#8592; gene on the &#39;reverse reading frame&#39;. &#8224;These changes are relative to a (A)10&#8594;11 frameshift present in the isolate of S. acidocaldarius DSM639 (Supplementary Table 1). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66271/Table 2.xlsx" target="_blank">Please click here to download this Table.</a>
Supplementary Table 1: Mutations present in the isolate of S. acidocaldarius DSM639 relative to the reference sequence (RefSeq accession NC_007181.1). &#8594; gene on the &#39;forward reading frame&#39;; &#8592; gene on the &#39;reverse reading frame&#39;. &#8225;SACI_RS04020 is annotated as a pseudogene in NC_007181.1, but the &#916;1 bp frameshift mutation observed here putatively restores its function as with the mutation, it encodes a protein with 100% identity to rgy reverse gyrase gene (RefSeq accession WP_176586667.1). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66271/Supplementary Table-1.zip" target="_blank">Please click here to download this File.</a>

Author Spotlight: Understanding Microbe Adaptation Using Innovative Techniques for Exploring Thermophilic Evolution

Here, we present an experimental evolution protocol for adaptation in thermophiles utilizing low-cost, energy-efficient bench-top thermomixers as incubators. The technique is demonstrated through the characterization of temperature adaptation in Sulfolobus acidocaldarius, an archaeon with an optimal growth temperature of 75 &#176;C.

Adaptation at the Extremes of Life: Experimental Evolution with the Extremophile Archaeon Sulfolobus acidocaldarius

The archaeon Sulfolobus acidocaldarius has emerged as a promising thermophilic model system. Investigating how thermophiles adapt to changing temperatures ...

adaptation-at-extremes-life-experimental-evolution-with-extremophile

Nanyang Technological University

Research

JoVE Journal

Biology

660 Views.  The University of Manchester. Het onderzoek in onze groep richt zich op het begrijpen van de regels van evolutie. Met dit project hebben we een nieuw protocol ontwikkeld om te bestuderen hoe thermofiele microben evolueren, met behulp van gecontroleerde laboratoriumexperimenten. Dit zal ons in staat stellen vragen te beantwoorden zoals hoe ze reageren op veranderingen in het milieu door middel van adaptieve evolutie.Een grote uitdaging is het beheersen van de teeltomstandigheden. Th...

Video: Auteur in de schijnwerpers: Aanpassing van microben begrijpen met behulp van innovatieve technieken voor het verkennen van thermofiele evolutie

Procedure for Adaptive Laboratory Evolution of Microorganisms Using a Chemostat

Natural evolution involves genetic diversity such as environmental change and a selection between small populations. Adaptive laboratory evolution (ALE) refers to the experimental situation in which evolution is observed using living organisms under controlled conditions and stressors; organisms are thereby artificially forced to make evolutionary changes. Microorganisms are subject to a variety of stressors in the environment and are capable of regulating certain stress-inducible proteins to increase their chances of survival. Naturally occurring spontaneous mutations bring about changes in a microorganism's genome that affect its chances of survival. Long-term exposure to chemostat culture provokes an accumulation of spontaneous mutations and renders the most adaptable strain dominant. Compared to the colony transfer and serial transfer methods, chemostat culture entails the highest number of cell divisions and, therefore, the highest number of diverse populations. Although chemostat culture for ALE requires more complicated culture devices, it is less labor intensive once the operation begins. Comparative genomic and transcriptome analyses of the adapted strain provide evolutionary clues as to how the stressors contribute to mutations that overcome the stress. The goal of the current paper is to bring about accelerated evolution of microorganisms under controlled laboratory conditions.

Here, we present a protocol to obtain adaptive laboratory evolution of microorganisms under conditions using chemostat culture. Also, genomic analysis of the evolved strain is discussed.

Natural evolution involves genetic diversity such as environmental change and a selection between small populations. Adaptive laboratory evolution (ALE) ...

Genetics

Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative medicine. A non-invasive method to monitor the transplanted stem cells repeatedly in vivo would greatly enhance our ability to understand the mechanisms that control stem cell death and identify trophic factors and signaling pathways that improve stem cell engraftment. MR imaging has been proven to be an effective tool for the in vivo depiction of stem cells with near microscopic anatomical resolution. In order to detect stem cells with MR, the cells have to be labeled with cell specific MR contrast agents. For this purpose, iron oxide nanoparticles, such as superparamagnetic iron oxide particles (SPIO), are applied, because of their high sensitivity for cell detection and their excellent biocompatibility. SPIO particles are composed of an iron oxide core and a dextran, carboxydextran or starch coat, and function by creating local field inhomogeneities, that cause a decreased signal on T2-weighted MR images. This presentation will demonstrate techniques for labeling of stem cells with clinically applicable MR contrast agents for subsequent non-invasive in vivo tracking of the labeled cells with MR imaging.

For the evaluation of new stem cell therapies it is important to non-invasively track the injected cells in vivo. This video will show you how to label human mesenchymal and embryonic stem cells with iron oxide based contrast agents in vivo for subsequent MR imaging in vivo.

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative ...

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo labeling of stem cells with a fluorescent dye, subsequent intravenous injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. The presented technique demonstrates how we label human mesenchymal stem cells (hMSC) by simple incubation with the lipophilic fluorescent dye DiD (C67H103CIN2O3S) and how we label human embryonic stem cells (hESC) with the FDA approved fluorescent dye Indocyanine Green (ICG). The uptake mechanism is via adherence and diffusion of the lypophilic dye across the phospholipid cell membrane bilayer. The labeling efficiency is usually improved if the cells are incubated with the dye in serum-free media as opposed to incubation in serum-containing media. Furthermore, the addition of the transfection agent Protamine Sulfate significantly improves contrast agent uptake.

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

Quantification of Plasmid-Mediated Antibiotic Resistance in an Experimental Evolution Approach

Plasmids play a major role in microbial ecology and evolution as vehicles of lateral gene transfer and reservoirs of accessory gene functions in microbial populations. This is especially the case under rapidly changing environments such as fluctuating antibiotics exposure. We recently showed that plasmids maintain antibiotic resistance genes in Escherichia coli without positive selection for the plasmid presence. Here we describe an experimental system that allows following both the plasmid genotype and phenotype in long-term evolution experiments. We use molecular techniques to design a model plasmid that is subsequently introduced to an experimental evolution batch system approach in an E. coli host. We follow the plasmid frequency over time by applying replica plating of the E. coli populations while quantifying the antibiotic resistance persistence. In addition, we monitor the conformation of plasmids in host cells by analyzing the extent of plasmid multimer formation by plasmid nicking and agarose gel electrophoresis. Such an approach allows us to visualize not only the genome size of evolving plasmids but also their topological conformation-a factor highly important for plasmid inheritance. Our system combines molecular strategies with traditional microbiology approaches and provides a set-up to follow plasmids in bacterial populations over a long time. The presented approach can be applied to study a wide range of mobile genetic elements in the future.

Our experimental approach provides a strategy to follow plasmid abundance and antibiotic resistance over time in bacterial populations.

Plasmids play a major role in microbial ecology and evolution as vehicles of lateral gene transfer and reservoirs of accessory gene functions in microbial ...

Bioprospecting of Extremophilic Microorganisms to Address Environmental Pollution

Geothermal springs are rich in various metal ions due to the interaction between rock and water that takes place in the deep aquifer. Moreover, due to seasonality variation in pH and temperature, fluctuation in element composition is periodically observed within these extreme environments, influencing the environmental microbial communities. Extremophilic microorganisms that thrive in volcanic thermal vents have developed resistance mechanisms to handle several metal ions present in the environment, thus taking part to complex metal biogeochemical cycles. Moreover, extremophiles and their products have found an extensive foothold in the market, and this holds true especially for their enzymes. In this context, their characterization is functional to the development of biosystems and bioprocesses for environmental monitoring and bioremediation. To date, the isolation and cultivation under laboratory conditions of extremophilic microorganisms still represent a bottleneck for fully exploiting their biotechnological potential. This work describes a streamlined protocol for the isolation of thermophilic microorganisms from hot springs as well as their genotypical and phenotypical identification through the following steps: (1) Sampling of microorganisms from geothermal sites ("Pisciarelli", a volcanic area of Campi Flegrei in Naples, Italy); (2) Isolation of heavy metal resistant microorganisms; (3) Identification of microbial isolates; (4) Phenotypical characterization of the isolates. The methodologies described in this work might be generally applied also for the isolation of microorganisms from other extreme environments.

The isolation of heavy metal-resistant microbes from geothermal springs is a hot topic for the development of bioremediation and environmental monitoring biosystems. This study provides a methodological approach for isolating and identifying heavy metal tolerant bacteria from hot springs.

Geothermal springs are rich in various metal ions due to the interaction between rock and water that takes place in the deep aquifer. Moreover, due to ...

Environment

Daily Transfers, Archiving Populations, and Measuring Fitness in the Long-Term Evolution Experiment with Escherichia coli

The Long-Term Evolution Experiment (LTEE) has followed twelve populations of Escherichia coli as they have adapted to a simple laboratory environment for more than 35 years and 77,000 bacterial generations. The setup and procedures used in the LTEE epitomize reliable and reproducible methods for studying microbial evolution. In this protocol, we first describe how the LTEE populations are transferred to fresh medium and cultured each day. Then, we describe how the LTEE populations are regularly checked for possible signs of contamination and archived to provide a permanent frozen "fossil record" for later study. Multiple safeguards included in these procedures are designed to prevent contamination, detect various problems when they occur, and recover from disruptions without appreciably setting back the progress of the experiment. One way that the overall tempo and character of evolutionary changes are monitored in the LTEE is by measuring the competitive fitness of populations and strains from the experiment. We describe how co-culture competition assays are conducted and provide both a spreadsheet and an R package (fitnessR) for calculating relative fitness from the results. Over the course of the LTEE, the behaviors of some populations have changed in interesting ways, and new technologies like whole-genome sequencing have provided additional avenues for investigating how the populations have evolved. We end by discussing how the original LTEE procedures have been updated to accommodate or take advantage of these changes. This protocol will be useful for researchers who use the LTEE as a model system for studying connections between evolution and genetics, molecular biology, systems biology, and ecology. More broadly, the LTEE provides a tried-and-true template for those who are beginning their own evolution experiments with new microbes, environments, and questions.

Daily Transfers, Archiving Populations, and Measuring Fitness in the Long-Term Evolution Experiment with Escherichia coli

This protocol describes how to maintain the Escherichia coli Long-Term Evolution Experiment (LTEE) by performing its daily transfers and periodic freeze-downs and how to conduct competition assays to measure fitness improvements in evolved bacteria. These procedures can serve as a template for researchers starting their own microbial evolution experiments.

The Long-Term Evolution Experiment (LTEE) has followed twelve populations of Escherichia coli as they have adapted to a simple laboratory environment for ...

In Situ Simulated Anaerobic Method for Culturing Environmental Microbes

A Set of In Situ Informed Simulated Medium Formats for Culturing Environmentally Acquired Anaerobic Microorganisms

Culture-dependent research of anaerobic microorganisms rests upon methodological competence. These methods must create and maintain suitable growth conditions (e.g., pH and carbon sources) for anaerobic microorganisms while also allowing samples to be extracted without compromising the artificial environment. To this end, methods that are informed by and simulate an in situ environment can be of great aid in culturing microorganisms from that environment. Here, we outline an in situ informed and simulated anaerobic method for culturing terrestrial surface and subsurface microorganisms, emphasizing anaerobic sample collection with minimal perturbation. This protocol details the production of a customizable anaerobic liquid medium, and the environmental&#160;acquisition and in vitro growth of anaerobic microorganisms. The protocol also covers critical components of an anaerobic bioreactor used for environmental simulations of sediment and anaerobic liquid media for environmentally acquired cultures. We have included preliminary Next Generation Sequencing data from a maintained microbiome over the lifespan of a bioreactor where the active culture dynamically adjusted in response to an experimental carbon source.

Author Spotlight: Unraveling the Mysteries of Terrestrial Anaerobic Microorganisms in Uncharted Environments by In Situ Culturing

The focus of this paper is to detail best practices for making media for fastidious anaerobic microorganisms acquired from an environment. These methods help manage anaerobic cultures and can be applied to support the growth of elusive uncultured microorganisms, the "microbial dark matter."

Culture-dependent research of anaerobic microorganisms rests upon methodological competence. These methods must create and maintain suitable growth ...

Techniques for the Evolution of Robust Pentose-fermenting Yeast for Bioconversion of Lignocellulose to Ethanol

Lignocellulosic biomass is an abundant, renewable feedstock useful for production of fuel-grade ethanol and other bio-products. Pretreatment and enzyme saccharification processes release sugars that can be fermented by yeast. Traditional industrial yeasts do not ferment xylose (comprising up to 40% of plant sugars) and are not able to function in concentrated hydrolyzates. Concentrated hydrolyzates are needed to support economical ethanol recovery, but they are laden with toxic byproducts generated during pretreatment. While detoxification methods can render hydrolyzates fermentable, they are costly and generate waste disposal liabilities. Here, adaptive evolution and isolation techniques are described and demonstrated to yield derivatives of the native Scheffersomyces stipitis strain NRRL Y-7124 that are able to efficiently convert hydrolyzates to economically recoverable ethanol despite adverse culture conditions. Improved individuals are enriched in an evolving population using multiple selection pressures reliant on natural genetic diversity of the S. stipitis population and mutations induced by exposures to two diverse hydrolyzates, ethanol or UV radiation. Final evolution cultures are dilution plated to harvest predominant isolates, while intermediate populations, frozen in glycerol at various stages of evolution, are enriched on selective media using appropriate stress gradients to recover most promising isolates through dilution plating. Isolates are screened on various hydrolyzate types and ranked using a novel procedure involving dimensionless relative performance index (RPI) transformations of the xylose uptake rate and ethanol yield data. Using the RPI statistical parameter, an overall relative performance average is calculated to rank isolates based on multiple factors, including culture conditions (varying in nutrients and inhibitors) and kinetic characteristics. Through application of these techniques, derivatives of the parent strain had the following improved features in enzyme saccharified hydrolyzates at pH 5-6: reduced initial lag phase preceding growth, reduced diauxic lag during glucose-xylose transition, significantly enhanced fermentation rates, improved ethanol tolerance and accumulation to 40 g/L.

Adaptive evolution and isolation techniques are described and demonstrated to yield derivatives of Scheffersomyces stipitis strain NRRL Y-7124 that are able to rapidly consume hexose and pentose mixed sugars in enzyme saccharified undetoxified hydrolyzates and to accumulate over 40 g/L ethanol.

Lignocellulosic biomass is an abundant, renewable feedstock useful for production of fuel-grade ethanol and other bio-products. Pretreatment and enzyme ...

Bioengineering

Automated Microbial Cultivation and Adaptive Evolution using Microbial Microdroplet Culture System (MMC)

Conventional microbial cultivation methods usually have cumbersome operations, low throughput, low efficiency, and large consumption of labor and reagents. Moreover, microplate-based high-throughput cultivation methods developed in recent years have poor microbial growth status and experiment parallelization because of their low dissolved oxygen, poor mixture, and severe evaporation and thermal effect. Due to many advantages of micro-droplets, such as small volume, high throughput, and strong controllability, the droplet-based microfluidic technology can overcome these problems, which has been used in many kinds of research of high-throughput microbial cultivation, screening, and evolution. However, most prior studies remain&#160;at the stage of laboratory construction and application. Some key issues, such as high operational requirements, high construction difficulty, and lack of automated integration technology, restrict the wide application of droplet microfluidic technology in microbial research. Here, an automated Microbial Microdroplet Culture system (MMC) was successfully developed based on droplet microfluidic technology, achieving the integration of functions such as inoculation, cultivation, online monitoring, sub-cultivation, sorting, and sampling required by the process of microbial droplet cultivation. In this protocol, wild-type Escherichia coli (E. coli) MG1655 and a methanol-essential E. coli strain (MeSV2.2) were taken as examples to introduce how to use the MMC to conduct automated and relatively high-throughput microbial cultivation and adaptive evolution in detail. This method is easy to operate, consumes less labor and reagents, and has high experimental throughput and good data parallelity, which has great advantages compared with conventional cultivation methods. It provides a low-cost, operation-friendly, and result-reliable experimental platform for scientific researchers to conduct related microbial research.

Automated Microbial Cultivation and Adaptive Evolution using Microbial Microdroplet Culture System MMC

This protocol describes how to use the Microbial Microdroplet Culture system (MMC) to conduct automated microbial cultivation and adaptive evolution. MMC can cultivate and sub-cultivate microorganisms automatically and continuously and monitor online their growth with relatively high throughput and good parallelization, reducing labor and reagent consumption.

Conventional microbial cultivation methods usually have cumbersome operations, low throughput, low efficiency, and large consumption of labor and ...

Design and Use of Multiplexed Chemostat Arrays

Chemostats are continuous culture systems in which cells are grown in a tightly controlled, chemically constant environment where culture density is constrained by limiting specific nutrients.1,2 Data from chemostats are highly reproducible for the measurement of quantitative phenotypes as they provide a constant growth rate and environment at steady state. For these reasons, chemostats have become useful tools for fine-scale characterization of physiology through analysis of gene expression3-6 and other characteristics of cultures at steady-state equilibrium.7 Long-term experiments in chemostats can highlight specific trajectories that microbial populations adopt during adaptive evolution in a controlled environment. In fact, chemostats have been used for experimental evolution since their invention.8 A common result in evolution experiments is for each biological replicate to acquire a unique repertoire of mutations.9-13 This diversity suggests that there is much left to be discovered by performing evolution experiments with far greater throughput. 
We present here the design and operation of a relatively simple, low cost array of miniature chemostats&mdash;or ministats&mdash;and validate their use in determination of physiology and in evolution experiments with yeast. This approach entails growth of tens of chemostats run off a single multiplexed peristaltic pump. The cultures are maintained at a 20 ml working volume, which is practical for a variety of applications. It is our hope that increasing throughput, decreasing expense, and providing detailed building and operation instructions may also motivate research and industrial application of this design as a general platform for functionally characterizing large numbers of strains, species, and growth parameters, as well as genetic or drug libraries.

We developed and validated a small-footprint array of miniature chemostats built from readily available parts for low cost. Physiological and experimental evolution results were similar to larger volume chemostats. The ministat array provides a compact, inexpensive, and accessible platform for traditional chemostat experiments, functional genomics, and chemical screening applications.

Chemostats are continuous culture systems in which cells are grown in a tightly controlled, chemically constant environment where culture density is ...

Adaptation at the Extremes of Life: Experimental Evolution with the Extremophile Archaeon Sulfolobus acidocaldarius

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