Automated Laboratory Growth Assessment and Maintenance of Azotobacter vinelandii

Azotobacter vinelandii (A. vinelandii) is a commonly used model organism for the study of aerobic respiration, the bacterial production of several industrially relevant compounds, and, perhaps most significantly, the genetics and biochemistry of biological nitrogen fixation. Laboratory growth assessments of A. vinelandii are useful for evaluating the impact of environmental and genetic modifications on physiological properties, including diazotrophy. However, researchers typically rely on manual growth methods that are oftentimes laborious and inefficient. We present a protocol for the automated growth assessment of A. vinelandii on a microplate reader, particularly well‐suited for studies of diazotrophic growth. We discuss common pitfalls and strategies for protocol optimization, and demonstrate the protocol's application toward growth evaluation of strains carrying modifications to nitrogen‐fixation genes. © 2021 The Authors.


INTRODUCTION
Azotobacter vinelandii (A. vinelandii), a Gram-negative, obligately aerobic, diazotrophic Gamma proteobacterium, has been an important model organism since its discovery more than a century ago (Lipman, 1903). One of the most significant contributions of the A. vinelandii model has been toward understanding the evolution, genetics, and biochemistry of nitrogenase-catalyzed nitrogen fixation (Dos Santos, 2019; Hoffman, Lukoyanov, Figure 2 Mean A. vinelandii DJ diazotrophic doubling times measured grown across a range of temperatures (left) and from preculture inocula of varying ages (right). Mean doubling times calculated across three experimental trials. Error bars represent 1σ. Asterisks indicate p-values ≤0.0001 calculated from a post-hoc Tukey's HSD test following a one-way ANOVA.
for development of this protocol, generously provided by Dennis Dean (Virginia Tech). However, other strains may be used depending on the desired application.

Optimization of growth conditions
Microplate readers with temperature control and continuous shaking capabilities can be used for A. vinelandii maintenance and growth rate determination. Standard A. vinelandii cultures are typically grown in flasks and shaken from 100 to 300 rpm at 30°C (Arragain et al., 2017;Dos Santos, 2019;McRose et al., 2017;Mus et al., 2017). However, these growth conditions can be further optimized or varied depending on experimental design and instrument capabilities.
Factors that may impact growth and should, thus, be considered when designing the experiment include temperature, shaking speed, culture volume, and inoculum preparation. As an example, we assessed A. vinelandii growth as a function of variation in temperature and preculture inoculum age (Fig. 2). We found that both temperature and preculture inoculum age led to significant (p < 0.05) differences in doubling times, with both increasing temperature and increasing inoculum age resulting in faster doubling times across the measured range. It is recommended that the user similarly optimize for key growth conditions with their equipment. Culture volume in particular may have a significant effect on growth rate by altering the amount of headspace in the well and culture movement during shaking. Culture volume should be optimized after shaking speed, as different volumes may be optimal at different shaking speeds. Additional care should be given to ensure consistency across the plate wells, which can be accomplished by using a lid or gas-permeable membrane to minimize evaporation and cross-contamination. The use of these items may result in slower A. vinelandii growth rates as compared to flask-grown cultures due to impeded aeration but should not preclude comparisons across samples grown in similar conditions.

Reproducibility
All qualitative and quantitative experimental parameters, particularly those used to operate the microplate reader, should be reported to facilitate future reproducibility (Chavez, Ho, & Tan, 2017). Multiple repeat measurements can be made simultaneously across aliquots of the same samples in a single microplate, but experimental replicates should be performed on different inoculations and days to account for variability of the bacterial stock and day-to-day instrument performance.

Data analysis
When analyzing growth measurements from high-throughput procedures, it is recommended to use reproducible and efficient data analysis methods. Several free Carruthers et al.

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PREPARATION OF A. VINELANDII PLATE CULTURES FROM FROZEN STOCKS
A growth rate experiment of A. vinelandii begins with strain recovery from frozen stocks. A. vinelandii stocks are typically kept in a 7% DMSO storage buffer and stored at -80°C. Frozen cells can then be directly streaked on solid medium to prepare isogenic plate cultures for subsequent growth rate assessments.
A. vinelandii is generally grown in Burk's medium (B medium; see Reagents and Solutions), which may be supplemented with a nitrogen source (BN medium), antibiotics, or modified trace metals as desired. This protocol describes recovery on BN plates (rather than B plates) so that cells can be grown regardless of the diazotrophic capabilities of the strain, which can later be assessed during the growth rate experiment. If needed, recovery can also be accomplished with BN plates supplemented with antibiotics.

PREPARATION OF A. VINELANDII LIQUID PRECULTURES
Preparation of A. vinelandii liquid precultures prior to the growth rate experiment is necessary to produce inocula that are in a consistent physiological state across different Carruthers et al.

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Current Protocols strains and replicates. The age of the preculture at the time of inoculation into the primary growth culture can be optimized as needed. A saturated preculture tends to provide a longer and more easily detectable lag phase for curve fitting in downstream growth rate analyses. Furthermore, it minimizes the volume of preculture needed to inoculate the primary culture, which can introduce undesirable residual medium additions (such as supplemental nitrogen) from the preculture that result in a variable lag phase.

AUTOMATED GROWTH RATE EXPERIMENT OF A. VINELANDII ON A MICROPLATE READER
This protocol focuses on the automated assessment of A. vinelandii diazotrophic growth in B (nitrogen-free) medium on a microplate reader. Liquid precultures grown in BN (Basic Protocol 2) are used to inoculate B medium to a desired starting optical density at 600 nm (OD 600 ) for the growth rate experiment. In B medium, A. vinelandii quickly use up any residual ammonia from the preculture and begin expressing nitrogenase genes for nitrogen fixation. Detected growth is, thus, directly related to the strain's ability to express nitrogenase and grow diazotrophically. Experiments targeting other aspects of A. vinelandii physiology can be designed by modifying the preculture and primary culture medium with, for example, antibiotics or different concentrations of trace metals. Over the course of the growth rate experiment, the microplate reader can provide automated temperature control, shaking, and optical density measurements, all parameters which can be varied to test different growth conditions as permitted by the instrument capabilities.

Materials
A. vinelandii saturated preculture in BN medium (from Basic Protocol 2) B medium (sterile; see recipe) Carruthers et al. 3. Measure the OD 600 of the diluted preculture on the microplate reader, taking a single absorbance reading of the wells.

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Current Protocols OD 600 measurement of the initial diluted preculture should be performed using the same microplate reader that will be used for the growth rate experiment. This removes the potential for measurement variation across different spectrophotometers and ensures that the investigator accurately prepares the primary growth culture to the desired optical density. Zeroing using the blank reference can be performed automatically by the plate reader, or the blank absorbance reading can simply be subtracted from the preculture reading to determine the actual OD 600 of the sample.
4. Based on the initial OD 600 value of the diluted preculture, in a glass flask (or other appropriate vessel), inoculate B medium to obtain a final OD 600 of ∼0.05 for the main growth culture. Swirl gently to mix.
Prepare a final volume that can sufficiently be aliquoted across the desired number of wells for the growth rate experiment. Note that excess volume may be needed to comfortably pipet from a reagent reservoir in the following step (e.g., for 125 μl across 96 wells, prepare >20 ml). Antibiotics can be added to the B medium prior to inoculation if desired.
Prepare microplate and perform growth rate experiment 5. Pour the primary growth culture into a reagent reservoir. Use a multichannel pipette to aliquot 125 μl across the wells of a microplate. Reserve at least one well to fill with 125 μl of B medium to serve as a blank reference for the experiment.
6. Use a lid or gas-permeable membrane to cover the wells of the microplate.
If using a lid, note that significant evaporation can still occur for wells close to the edge of the microplate and it may be preferable to leave these wells empty (Chavez et al., 2017;Hall et al., 2014). If using a membrane, take care to ensure that there are minimal creases or bubbles when applied. Improper membrane application may lead to inconsistent growth rates across the microplate because these surface area differences influence aeration.
7. Insert the prepared microplate into the reader, set the instrument parameters, and start the growth experiment program.
Microplate reader parameters that should be considered include: • 8. Save the growth experiment file and export for subsequent data analysis (see Strategic Planning and Fig. 4).

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Current Protocols Figure 4 Diazotrophic growth curves for wild-type (WT) and engineered A. vinelandii strains. Optical density at 600 nm (OD 600 ) measured once per hour for multiple wells in a single experimental trial. Mean growth curve for each strain indicated by solid line.

Burk's (B) medium/Burk's nitrogen-supplemented (BN) medium, solid
Weigh 8 g agar (Sigma-Aldrich, cat. no. A1296) into a 1-L bottle Dilute 4.5 ml 100× phosphate buffer into 445 ml dH 2 O Sterilize by autoclaving for 20 min at 121°C Cool to 55°C in a water bath Add 50 ml sterile 10× salts solution Optional for BN medium: Add 5 ml sterile 100× ammonium acetate solution Pour ∼25 ml into plates and cool Store plates in sealed sleeve up to several months at 4°C

Phosphate buffer, 1×
Dilute 5 ml 100× phosphate buffer into 495 ml dH 2 O Sterilize by autoclaving for 20 min at 121°C Store up to 1 year at room temperature

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The aerobic A. vinelandii is an appealing model organism for laboratory work due to its relative ease of maintenance, genetic tractability, and non-pathogenicity (Biosafety Level 1), as compared with other pathogenic or anaerobically cultivated diazotroph models (e.g., Clostridium pasteurianum, Klebsiella pneumoniae, Rhodopseudomonas palustris). A. vinelandii can be maintained in ambient, benchtop conditions, though growth is typically optimized and standardized in an in-cubator. Natural competency for transformation in A. vinelandii can be induced via metal starvation and visually confirmed due to the associated production of fluorescent green siderophores (McRose et al., 2017). This characteristic makes it a highly suitable organism for genetic manipulation, as reviewed in (Dos Santos, 2019). Growth rate assessments of genetically modified A. vinelandii can, therefore, reveal the physiological contributions of nitrogenase and nitrogenase-related genes (Arragain et al., 2017;Garcia, McShea, Kolaczkowski, & Kaçar, 2020;McRose et al., 2017;Mus, Colman, Peters, & Boyd, 2019;Plunkett et al., 2020).

Critical Parameters and Troubleshooting
It is important to optimize conditions for A. vinelandii growth on a microplate reader prior to growth rate assessment to ensure consistency and reproducibility across biological and technical replicates (see Strategic Planning). Growth conditions that should be considered include temperature, shaking speed, culture volume, and inoculum preparation.
A common problem encountered during growth rate assessment on a microplate reader (Basic Protocol 3) is well evaporation over the approximately 48 to 72 hr needed for A. vinelandii cultures to reach saturation. Evaporation can be minimized by use of a lid or gas-permeable membrane. However, significant evaporation can still occur at plate edges when using a lid (Chavez et al., 2017), and improper application of the membrane can result in variable growth rates across the plate. Since such variability is challenging to eliminate entirely, it is important to include several technical replicates across the plate and avoid measuring wells at the plate edges when using a lid.

Time Considerations
The initial recovery of Azotobacter strains (Basic Protocol 1) and preparation of isogenic plate cultures (Basic Protocol 2) takes approximately 6 days. Preculture preparation (Basic Protocol 2) takes approximately 24 hr. This time can be optimized but should be made consistent across replicates. Each microplate reader growth experiment takes approximately Carruthers et al.

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Understanding results
This protocol can also be used to compare the growth rates and characteristics of different wild-type and engineered A. vinelandii strains, as well as different physical and nutritional growth conditions. To provide an example of anticipated results for the protocol described here, we conducted diazotrophic growth rate experiments on wild-type Azotobacter vinelandii DJ and three strains harboring modifications to the nitrogenase nifD gene. The nifD gene encodes the active sitecontaining subunit of the nitrogenase enzyme. Modifications to this gene are expected to influence nitrogenase N 2 -reduction and, thus, the ability of A. vinelandii to grow diazotrophically. The modified strains include "AK013" and "AK014", which have 93% and 81% nifD DNA identity to nifD of the wild-type DJ strain, respectively, and a DJ nifD deletion strain. Figure 4 shows growth curves for each A. vinelandii strain, and Table 1 reports mean doubling times calculated with the R package GrowthCurver (Sprouffske & Wagner, 2016). We did not detect diazotrophic growth for DJ nifD. For Trials 1 and 2, AK014 grew slower than both DJ and AK013 (p < 0.05; calculated from a post-hoc Tukey's HSD test following a one-way ANOVA), but no difference in doubling times was found for DJ and AK013. However, for Trial 3, DJ grew significantly slower than both AK013 and AK014. This result highlights the need to repeat growth experiments on multiple days to account for day-to-day instrument variability. This automated protocol for evaluating diazotrophic growth differences across different A. vinelandii strains can be adapted for a variety of additional applications.