A Review of Methods To Determine Viability, Vitality, and Metabolic Rates in Microbiology
A Review of Methods To Determine Viability, Vitality, and Metabolic Rates in Microbiology
A Review of Methods To Determine Viability, Vitality, and Metabolic Rates in Microbiology
Viability and metabolic assays are commonly used as proxies to assess the overall
metabolism of microorganisms. The variety of these assays combined with little
information provided by some assay kits or online protocols often leads to mistakes
Edited by:
Frank Schreiber,
or poor interpretation of the results. In addition, the use of some of these assays
Federal Institute for Materials is restricted to simple systems (mostly pure cultures), and care must be taken in
Research and Testing (BAM),
their application to environmental samples. In this review, the necessary data are
Germany
compiled to understand the reactions or measurements performed in many of the
Reviewed by:
Helmut Maske, assays commonly used in various aspects of microbiology. Also, their relationships to
Center for Scientific Research each other, as metabolism links many of these assays, resulting in correlations between
and Higher Education in Ensenada
(CICESE), Mexico
measured values and parameters, are discussed. Finally, the limitations of these assays
Roland Hatzenpichler, are discussed.
Montana State University,
United States Keywords: metabolism, viability, assay, redox dyes, electron acceptors, carbon sources
*Correspondence:
Olivier Braissant
Olivier.braissant@unibas.ch
INTRODUCTION
Specialty section:
Metabolism can be defined as the sum of all reactions in a living organism aimed at maintenance,
This article was submitted to development, and reproduction (division for microbes). Consequently, measuring all reactions that
Microbial Physiology and Metabolism, contribute to metabolism in microbial cells is impossible with current tools even if metabolomics
a section of the journal using NMR and mass spectrometry (MS) can provide very valuable snapshots of the metabolites
Frontiers in Microbiology present (Emwas et al., 2013; Alonso et al., 2015; Markley et al., 2017; Zang et al., 2019). In addition,
Received: 08 April 2020 very few methods allow dynamic measurement of metabolism. Therefore, for many purposes
Accepted: 08 October 2020 ranging from environmental sciences to medical microbiology, microbial metabolism is usually
Published: 17 November 2020 assessed by using proxies that focus on different aspects of the process. Among these so-called
Citation: metabolic assays, some are nonspecific proxies focusing on various types of metabolism [such as
Braissant O, tetrazolium reduction, fluorescein diacetate (FDA) hydrolysis, or calorimetry], while some assays
Astasov-Frauenhoffer M, Waltimo T are more focused on and limited to specific types of metabolism. Indeed, one can assess metabolism
and Bonkat G (2020) A Review
using electron acceptor consumption (O2 , NO3 − , SO4 2− , and others) or the production of their
of Methods to Determine Viability,
Vitality, and Metabolic Rates
reduced form (NO2 − and H2 S, for example). Similarly, the consumption of carbon sources and
in Microbiology. production of by-products (CO2 , fermentation products, and others) are also valuable tools.
Front. Microbiol. 11:547458. As these assays all focus on metabolism, there are correlations between some of them, making
doi: 10.3389/fmicb.2020.547458 comparison or validation possible.
Metabolic assays have been used for a wide variety of the measure of a proxy in the form of a concentration is still a
applications. For example, in ecological studies, such assays proxy for a rate.
can be used to evaluate the number of active bacteria in a
sample (microbial mat, food sample, or other). However, in
pharmacological and biotechnological applications, these assays REDOX ASSAYS
are less often focused on metabolic rates and are mostly used
to assess viability to compare the effects of different products Tetrazolium Salts
on microbial cultures. Similarly, yield and other important Tetrazolium salts represent a large family of compounds
information can be gathered from some of these assays or (Table 1) that can be used to measure redox activity in
their combinations. metabolically active cells (Smith and McFeters, 1997; Berridge
Nevertheless, depending on the intended application et al., 2005; Grela et al., 2018; Stockert et al., 2018).
parameters, such as volume and sensitivity, the necessary In particular, tetrazolium reduction is associated with a
equipment can be a significant factor in the choice of a metabolic functional and active electron transport system (ETS). Colorless
assay. For example, metabolic assays with pure cultures can have tetrazolium salts pass the outer membrane of most tested
very different requirements from those with environmental or bacterial cells readily and are then reduced to different red to
food samples. Indeed, many of these assays have been devised violet formazan derivatives by reduced nicotinamide adenine
for pure cultures and are applicable only to such cultures. Many dinucleotides (NADH) or their phosphorylated derivative
have been modified for applicability to environmental studies, (NADPH)-dependent oxidoreductases and dehydrogenases of
but the resulting limitations are often neglected. In addition, the metabolically active cells (Figure 1). There are very few data
cost and practicality can also vary a lot, thus influencing potential on the ability of archaea to reduce tetrazolium salts, still some
application. Therefore, it is important to take these parameters pure cultures of Haloferax volcanii, Haloarcula marismortui,
into account and review the different assays. and Halobacterium sodomense have been shown to perform
such reduction (Oren, 1995). However, it must be noted that
tetrazolium might not be able to penetrate a large fraction of
CONFUSION BETWEEN AMOUNT AND fungal and other eukaryotic microbes to reach the mitochondria
ACTIVITY: A BRIEF WARNING and thus the active ETS or other enzymes able to reduce it
(Kumar and Tarafdar, 2003). Usually, it is considered that sites
Using all the techniques described below, it is crucial to where reactions occur along metabolic pathways are, in most
discriminate between activities (i.e., rates) and amounts (values cases, well-known and that as a consequence of their connection
measured at defined time points). In many studies, this difference with the ETS, the reactions are correlated with the respiration
is neglected, which results in directly linking growth to metabolic rate even under anaerobic conditions (Fontvieille et al., 1992).
activity. Many studies have measured metabolic activity using Some of the resulting formazan derivatives are soluble, while
growth (often measured by a net increase in cell number) as a others are not. Insoluble formazan compounds formed inside
proxy. Although such approximation can be understood as high the cells can be extracted with organic solvents (methanol,
metabolic activity is required for growth, it has major flaws. For ethanol, acetone, or other appropriate organic solvent mixtures)
example, in batch liquid culture, when entering the stationary to get a quantitative assessment of the amount produced. Several
phase, the cell number still increases while the metabolic activity parameters have been considered in optimizing the assay. Among
is often already decreasing. In this context, it is often worth them, final concentration of tetrazolium salts (enzyme saturation
coupling both measures and determining the activity per cell vs. dye toxicity), incubation time, added substrates (if any),
over the course of the culture. Ultimately, at the end of the pH of the assay, and storage conditions of the sample are
experiment, usually at the late stationary phase, there is a very important. Also, it is important to assess the presence of abiotic
large number of cells, but the overall metabolic activity is very reductant, which can trigger abiotic formation of formazan
low. On the other extreme of the spectrum, it was recently without active biological activity. Several studies have pointed
shown that even with no net biomass increase, biofilm could out that flavonoid, plant extract, or another reducing agent
have a very high metabolic activity as measured by calorimetry might lead to a significant reduction of tetrazolium dyes even
(Solokhina et al., 2017). This is also true at the community in formaldehyde-fixed samples. For this, it is recommended to
level; for example, in microbial mats, low “apparent” growth perform a blank measurement on using a sample fixed by adding
rates are observed; however, these microbial structures have been formaldehyde to a final concentration of 1.5% (Braissant et al.,
shown to have productivity and turnover equivalent to rain forest 2009). Note that a final concentration of formaldehyde up to 4.0%
(Guerrero and Mas, 1989; Briand et al., 2004). Currently, to assess can be used. Similarly, the presence of superoxide might also
metabolic activity, it is crucial to measure rates with a sufficient affect the results (Gong, 1997; Wieder et al., 1998; Créach et al.,
time resolution to allow determination of changes in the rates. 2003; Berridge et al., 2005; Wang et al., 2011; Grela et al., 2018).
It is also crucial that authors pay attention to their use of the Tetrazolium reduction is considered to be proportional not
terms “metabolism” and “metabolic activity” in their reports and only to the number of cells present but also to their metabolic
provide a proper explanation of whether rates or amounts (as activity. Indeed, inactive cells (even in large numbers) will
proxies) are being considered. Overall, the take-home message have minimal (if any) reduction activity. This can be used to
from this section is that metabolism always implies a rate, and determine the respective proportion of active and inactive cells
Short name Full name Tetrazolium Absorb. coef. (reduced Redox Formazan
solubility formazan) cm−1 ·M−1 intermediate solubility
TTC Triphenyl tetrazolium chloride 50 mg/ml 14,320 (sigma) Not required Insoluble
MTT Thiazolyl blue tetrazolium bromide 5 mg/ml 13,000–16,900 (578 nm) Not required Insoluble
INT Iodonitrotetrazolium chloride 4 mg/ml 12,000 (480–490 nm) Not required Insoluble
CTC 5-Cyano-2,3-di-(p-tolyl)tetrazolium chloride 15 mg/ml NA Not required Insoluble
Fluorescent compound
NBT Nitroblue tetrazolium 10 mg/ml 12,300 (580 nm) Not required Insoluble
XTT 2,3-Bis(2-methoxy-4-nitro-5-sulfophenyl)-2H- 2.5 mg/ml 21,600–23,800 Recommended Soluble
tetrazolium-5-carboxanilide inner salt (470–475 nm)
MTS MTS(5-(3-carboxymethoxyphenyl)-2(4,5,- 2.0 mg/ml 26,900 (490 nm) Recommended Soluble
dimethyl- thiazolyl)-3-(4 sulfophenyl)tetrazolium
inner salt
WST-1 2-(4-Iodophenyl)-3- (4-nitrophenyl)-5-(2,4- 10 mg/ml 37,000 (438 nm) Required Soluble
disulfophenyl)-2H-tetrazolium
WST-3 2-(4-Iodophenyl)-3-(2,4-dinitrophenyl)-5-(2,4- 10 mg/ml 30,000 (433 nm) Required Soluble
disulfophenyl)-2H-tetrazolium
WST-8 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)- 50 mg/ml (H2 O) 30,7000 (460 nm) Required Soluble
5-(2,4-disulfophenyl)-2H-tetrazolium 10 mg/ml (DMSO)
Data were compiled from the following references and websites: Sutherland and Learmonth (1997); Berridge et al. (2005); Grela et al. (2018); www.sigmaaldrich.com,
www.interchim.com, www.medchemexpress.com, www.abcam.com.
DMSO, dimethyl sulfoxide; NA, not applicable.
FIGURE 1 | Example of reduction of triphenyl tetrazolium into formazan. Note that many other derivatives of tetrazolium exist (Table 1).
in various samples by comparison to microscopy count, for especially for environmental studies. Several tetrazolium
example (Dufour and Colon, 1992; Haglund et al., 2002; Bartosch salts [especially 5-cyano-2,3-di-(p-tolyl)tetrazolium chloride
et al., 2003). Similarly, studies have shown that the production (CTC), but also iodonitrotetrazolium chloride (INT) and
of formazan per cell increases following an increase in growth 2,3- bis(2-methoxy-4-nitro-5-sulfophenyl)-2h-tetrazolium-5-
rate (Villegas-Mendoza et al., 2019). On the contrary, at a later carboxanilide inner salt (XTT); Table 1] have been shown to
stage of batch culture (late stationary phase), where most cells be toxic to some bacteria (Ullrich et al., 1996; Servais et al.,
are not growing anymore, staining of such cells by tetrazolium 2001; Hatzinger et al., 2003; Nielsen et al., 2003b; Villegas-
was poor (Fukui and Takii, 1989). Also, it must be noted Mendoza et al., 2015). Similarly, some viable and culturable
that different tetrazolium salts might be reduced differently, bacteria may not incorporate formazan derived from CTC or
making comparisons between studies difficult (Trevors, 1984). INT, even after long incubation times, thus emphasizing that
This adds to the complexity and needs to be clarified when using, some bacterial strains lack the ability to reduce tetrazolium
comparing, or reviewing tetrazolium studies data. to formazan. In environmental studies, it was shown that in
In addition, there are other limitations to the use of some samples, unlabeled (unstained) bacteria could still be
tetrazolium-based dyes that must be taken into account, responsible for a large fraction of the metabolic activity observed
(Servais et al., 2001). Therefore, in order to improve the results, with milder heating (55◦ C for 10 min) allows selection of the
the staining kinetics and tetrazolium concentration must be forms that will be assayed. However, proper neutralization of the
optimized for different environments and cell types (Servais added acid or base needs to be performed (Wagner and Scott,
et al., 2001). Also, this implies that tetrazolium reduction for 1994; Kern et al., 2014).
the estimation of environmental sample microbial activity is The assay is convenient and easy to perform (especially when
indicative of the most active microbial populations, whereas using a commercial kit). However, due to the reactivity of the
the contribution of fungal and other eukaryotic microbes to the NAD+ and NADP+ extracts, it is recommended to store them
measurement of dehydrogenase activity is extremely limited at −80◦ C after extraction for a maximum of 1 week, or they
(Kumar and Tarafdar, 2003). can be stored on ice for 1 h. Similarly, for complex (food or
Finally, depending on the studies considered, reduction environmental) samples, attention must be paid to the presence
of tetrazolium dyes is reported either as rates of reduction of inhibitory compounds that might alter the results of the
(i.e., moles of formazan produced per unit of time, and enzymatic assay. Furthermore, the complete buffer should be
eventually per mass or volume of sample) or as amount prepared fresh for each measurement. However, commercial
of formazan released [i.e., optical density (OD) or moles assays have procedures that simplify the preparation steps
of formazan]. Again, it is important to compare rates with and make the assay very reproducible under ideal conditions
other matching rates, for example, formazan production rates (see assay leaflet from producer’s website). Still comparing
with oxygen consumption rates (see example in Martínez- samples spiked with NAD+ , NADP+ , NADH, or NADPH
García et al., 2009; Maldonado et al., 2012). Similarly, amounts with unspiked samples might allow determining whether a
such as cell number (plate or microscopy count) should be commercial kit is usable under specific conditions, in particular
compared to the amount of formazan produced (see example in for complex samples.
Oren, 1987).
Resazurin (alamarBlueTM )
Resazurin (7-hydroxy-3H-phenoxazin-3-one 10-oxide) has
Using Tetrazolium Derivatives to Assess been used in the composition of many media for anaerobes
NAD+ , NADH, NADP+ , and NADPH because of its blue-violet color to indicate the presence of
Nicotinamide adenine dinucleotides (NAD+ /NADH) and their oxygen, which transitions to colorless when no dissolved
phosphorylated derivatives (NADP+ /NADPH) are important oxygen is available. Resazurin is also used for metabolic
redox-active molecules used in many catabolic and anabolic and viability assays (Liu, 1983; Lall et al., 2013; Heller
reactions. Ratios of NAD+ to NADH and NADP+ to NADPH and Edelblute, 2018). Similar to tetrazolium salts, the
are therefore considered key indicators of the overall intracellular dye is reduced by NAD(P)H-dependent oxidoreductases
redox potential and metabolic state. Because tetrazolium salts and dehydrogenases (Figure 3). Due to the chromogenic
are often used to assess the activity of NADH or NADPH and fluorogenic properties of its oxidized form (resazurin)
utilizing enzymes (Figure 1), it is also possible to assess the and reduced form (resorufin: 7-hydroxy-3H-phenoxazin-
amount of NAD+ , NADH, NADP+ , and NADPH using the 3-one), resazurin can be used for both types of assay.
redox nature of tetrazolium salts coupled with an appropriate Assays can be performed colorimetrically, as the resazurin
enzymatic system (substrate and enzyme) and electron mediator changes from blue to violet (molar absorption coefficient of
(redox intermediate) (Figure 2) (Kern et al., 2014; Spaans et al., resorufin = 54,000–58,500 cm−1 ·M−1 ). Similarly, resazurin
2015; Veskoukis et al., 2018). The assay is usually performed on is only weakly fluorescent as its irreversible reduction to
tissue or microbial pellet extracts. resorufin makes it highly fluorescent, thus allowing very sensitive
For NADP+ and NADPH, the assays rely on the conversion detection with a fluorometer. For this purpose, resazurin has
of glucose-6-phosphate (G6-P) to 6-phosphogluconolactone by been sold in assay kits under different trademarks, such as
glucose-6-phosphate dehydrogenase (G6PD; EC: 1.1.1.49), where alamarBlue, PrestoBlue, and UptiBlue, among others. However,
the simultaneous reduction of NADP+ to NADPH is coupled it must be noted that such commercial kits are only tested on
to the reduction of a tetrazolium salt [often thiazolyl blue standard microorganisms (such as Escherichia coli, Bacillus
tetrazolium bromide (MTT) or tetrazolium-based dye (WST); subtilis, Staphylococcus aureus, Candida albicans) and standard
Table 1] through a redox intermediate [phenazine ethosulfate mammalian cell lines. Therefore, their application to the study
(PES)] (Figure 2). Similarly, for the determination of NAD+ of nonstandard microorganisms and environmental samples
and NADH, the assays use ethanol dehydrogenase (ADH; must be validated/calibrated by the experimenters before use (see
EC: 1.1.1.1), which catalyzes the conversion of ethanol to examples below).
acetaldehyde. In this reaction, the reduction of NAD+ to NADH For environmental conditions and with pure culture,
is also linked to the reduction of a tetrazolium salt to its formazan resazurin reduction has been shown to be highly correlated
derivative through PES (Figure 2) (Wagner and Scott, 1994; to oxygen consumption (Liu, 1983; McNicholl et al., 2007;
Kern et al., 2014). González-Pinzón et al., 2012). Indeed, resazurin assays work
Oxidized (NAD+ , NADP+ ) and reduced (NADH, NADPH) best for aerobic or microaerophilic microorganisms. For
forms can be assessed separately, as the oxidized forms are easily example, Clostridium butyricum (a strict anaerobe) does not
destroyed by heating at 60◦ C for 30 min. Alternatively, alkaline reduce resazurin (Karakashev et al., 2003) like other strictly
(NaOH 0.2 M) or acidic (HCl 0.2 M) treatment in combination anaerobic Clostridium are reported to reduce tetrazolium salts
FIGURE 2 | Possible use of tetrazolium assay to assess the metabolic state of cells using ratios of nicotinamide adenine dinucleotides (NAD+ -NADH) or their
phosphorylated derivatives (NADP+ -NADPH). ADH, alcohol dehydrogenase; G6PD, glucose-6-phosphate dehydrogenase.
(List et al., 2019), maybe through other processes. Furthermore, ADENOSINE TRIPHOSPHATE ASSAYS
we can safely assume that resazurin is not likely to be reduced
by anaerobes (or during anaerobic respiration, such as sulfate Adenosine triphosphate (ATP) is a crucial molecule in
or nitrate reduction), as it is used in media as an oxygen redox microorganisms because of its role as a universal energy
indicator. This makes the application of resazurin for assays currency. However, it must be noted that ATP on its own can
a bit more limited compared to tetrazolium salts. Still, when only reveal a tiny fraction of the energetic state of a cell or a
looking at an aerobic (or microaerobic) setup, resazurin is a system as other adenylates [adenosine diphosphate (ADP) and
very sensitive assay, especially when used with fluorometry. adenosine monophosphate (AMP)] have to be accounted for (see
Another limitation to the use of resazurin is its toxicity toward below). When considering only ATP, the general assumption is
certain cells. Indeed, the toxicity is likely to be low for most that microorganisms generate ATP through catabolic reactions
microbes as the concentration of resazurin in the assay (0.4– and subsequently use it for “housekeeping,” growth, and
4 mM) is lower than the concentrations at which inhibition is replication. As a result, ATP is believed to indicate the presence
observed for some bacteria (Schmitt et al., 2013). Still, some of viable microorganisms in samples. Alternatively, as the
studies have mentioned its toxicity toward bacteria present in intracellular concentration of ATP per cell has been shown to
raw milk, pathogens, and human cell lines, especially tumor vary with changing environmental and physiological conditions,
cell lines (Ramsdell et al., 1935; Pace and Burg, 2013; Schmitt it can be used as a proxy to indicate the metabolic activity of cells
et al., 2013). Finally, the presence of nanomaterials has been (Schneider and Gourse, 2004; Mempin et al., 2013; Nescerecka
shown to influence resazurin reduction and its fluorescence, et al., 2016). Indeed, in cells losing viability, the ability to
and much care has to be taken in the presence of such materials synthetize ATP is lost and many biochemical reactions including
(Breznan et al., 2015). the action of ATPases rapidly deplete any remaining ATP
from the cytoplasm. In growing (viable) E. coli and Salmonella and production of AMP. The overall reaction (Figure 4) releases
typhimurium, the intracellular concentration of ATP varies from light that can be measured by a luminometer. Many assay kit
1 to 5 mM (Mempin et al., 2013). For a wider range of bacteria, reagents contain a detergent to allow cell lysis and recovery
values are between 0.1 and 26 fg ATP·CFU−1 (Kodaka et al., of intracellular ATP, as well as ATPase inhibitors to avoid loss
1996; Venkateswaran et al., 2003); however, the average is usually of ATP during the processing. Still, for very “dirty” or some
around 1 fg ATP·CFU−1 . The values can be converted to mM by environmental samples, care has to be taken that impurities do
assuming a cytoplasm volume1 of 0.67 µm3 ; however, one has not contain other compounds that might also affect the level of
to note that cell volume varies a lot between microbial species ATP extracted and/or measured (see below as well). This assay
and within the same species with varying growth conditions. The is by far the most popular ATP assay used. At the end of this
values for Bacillus species spores are much lower, ranging from assay, remaining adenylates (ADP and AMP) can be quantified
0.01 to 0.0002 fg ATP·CFU−1 ; however, in Bacillus spores, rather by converting them enzymatically into ATP using pyruvate kinase
high levels of 3-phosphoglyceric acid (3PGA), a potential rapid (EC 2.7.1.40 for ADP) or pyruvate kinase and adenylate kinase
source of ATP, are found (Ghosh et al., 2015; Setlow and Johnson, (also called myokinase; EC 2.7.4.3 for AMP) (Lee and Colston,
2019). Finally, for the yeast C. albicans, it is much higher, 213 fg 1986; Kinniment and Wimpenny, 1992) and performing an
ATP·CFU−1 . Of note, recent progress in single-cell imaging has additional luminescence measurement. The values can be used
shown that within the same culture, there was a fair amount of to calculate AEC (see above). Commercial kits are available for
variation (from less than 0.5 to 14 mM) in the distribution of ATP determination of ATP only or sequential determination of all
concentration within individual cells (Yaginuma et al., 2014). three adenylates.
Furthermore, the release of extracellular ATP has to be taken Colorimetric alternatives are also available. Among the
into account when assessing metabolism or viability, as a large alternatives, glycerol phosphorylation reaction can be used to
percentage of the ATP in a culture can be extracellular, especially assess the amount of ATP present. Glycerol added in excess
in the exponential growth phase or when exposed to disinfectants is phosphorylated by glycerol kinase (EC: 2.7.1.30) to produce
such as chlorine (Mempin et al., 2013; Nescerecka et al., 2016). glycerol-3-phosphate and ADP. The glycerol-3-phosphate is then
Therefore, ATP values have to be taken with care, especially further oxidized by glycerol phosphate oxidase (EC: 1.1.3.21),
as their use is still debated for some applications. Indeed, the producing dihydroxyacetone phosphate (glycerone phosphate)
interpretation of ATP measurements, especially with respect to and hydrogen peroxide (H2 O2 ). Finally, a peroxidase (EC:
viability and activity, can be improved if the other adenylates 1.11.1.7) catalyzes the redox-coupled reaction of H2 O2 with
(ADP and AMP) are taken into account (or other parameters 4-aminoantipyrine (4-AAP) and N-ethyl-N-(3-sulfopropyl)-m-
such as biomass or cell count). The concept of adenylate energy anisidine (ESPA), leading to the formation of a purple dye
charge (AEC) was introduced by Atkinson (1969) to reflect the that is measured at 540 or 570 nm (Figure 5). Again, correct
fact that metabolic processes are sensitive to levels of individual extraction of ATP and use of detergent are important to
adenine nucleotides or their relative proportions. AEC is defined recover ATP.
as [(ATP) + 0.5 (ADP)]/[(ATP) + (ADP) + (AMP)]. However, The advantage of the ATP assay is that you do not have
in some studies, it is simplified as (ATP)/[(ATP) + (ADP)]; thus, to rely on an incubation step with a population of viable cells
it is worth checking which one is used for comparison purposes. to convert a substrate (such as tetrazolium, resazurin, or FDA)
Still, it is often considered that the ratio of ATP, ADP, and AMP into a colored compound. In addition, for some applications
is functionally more relevant than the absolute concentration (mostly with cultures), there is also no need to remove cell culture
of ATP alone (De La Fuente et al., 2014). With respect to the medium or wash cells before adding the reagent, which can be
value of AEC, it is generally considered that metabolically active fully automated for high throughput. Still, serial measurements
and/or growing cells have AEC values between 0.70 and 0.95 are needed to assess the flow of intracellular and extracellular
(Lee and Colston, 1986; De La Fuente et al., 2014). Similarly, ATP and get a complete image of metabolic processes over time.
cells remain viable with AEC values of 0.5–0.8. Finally, in stress If sample processing needs to be performed at a later stage
conditions, AEC falls below 0.5, and values below 0.5 are usually (i.e., after all samples have been collected), it is recommended
considered to be incompatible with maintaining the minimal to snap-freeze the samples in liquid nitrogen or on dry ice.
level of homeostasis required for viability (Chapman et al., 1971). For this assay, ATP extraction remains a crucial step and
Still, Bacillus endospores can have an AEC value of 0.08, for different extraction methods were tested on microbial culture
example (Kodaka et al., 1996). AEC has been subjected to intense samples, showing strong variation in ATP recovery (Lundin
discussions and is considered as “misleadingly simplistic.” and Thore, 1975; Prioli et al., 1985; Stanley, 1989). Still, a
Still, many authors agree that it can be a valuable approach comparison between commercial luciferase-based ATP assays
to assess the broad homeostatic feature of ATP-utilizing and and 31P NMR ATP determination (which does not require an
ATP-replenishing reactions (Purich and Allison, 1999). extraction step) on blood cells did not show significant differences
Measurement of ATP can be performed using two types of (Marjanovic et al., 1993). For environmental samples, however,
commercial enzymatic assays. The first ones rely on the use of the determination of ATP is more difficult due to the poor
luciferase (firefly luciferase EC 1.13.12.7 or other luciferase) to recovery rate of some extraction methods (Karl and Holm-
oxidize luciferin into oxyluciferin with concomitant use of ATP Hansen, 1978; Webster et al., 1984) and potential hydrolysis
or other chemical interactions (Karl and Holm-Hansen, 1978;
1
https://bionumbers.hms.harvard.edu/ Karl, 1993). In addition, early studies on marine samples using
FIGURE 4 | Simplified reactions occurring during ATP assay using firefly luciferase.
charcoal columns found that it is likely that part of the ATP is unlike tetrazolium salt or resazurin reduction, FDA hydrolysis is
hydrolyzed during extraction and recovery. It is also mentioned not expected to be directly linked to O2 consumption, although
that filtration-induced metabolic stress might lead to a decrease in many studies have shown that the two are often correlated
ATP (however, total adenylate content remains stable). The exact (Fontvieille et al., 1992). Still, the assay can be widely used
process leading to ATP hydrolysis remains unclear; still, much in many setups as FDA hydrolysis capacity is widespread
care has to be taken when analyzing environmental samples (Schnurer and Rosswall, 1982; Gaspar et al., 2001; Prosser et al.,
and drawing conclusions about activity (or biomass). Also, for 2011; Liang et al., 2019; Long et al., 2019; Braun et al., 2020).
all types of samples, the pH must be controlled with care, as Intracellular hydrolysis of FDA results in the accumulation
it has been shown to affect the results (Posimo et al., 2014; of fluorescein (which is unable to pass cell membranes) in
Šimèíková and Heneberg, 2019). metabolically active cells. As a consequence, FDA hydrolysis
has often been used in combination with ethidium bromide or
propidium iodide penetration in damaged cells and binding to
FLUORESCEIN DIACETATE AND DNA in microscopic assays of viability (see review in Tawakoli
DERIVATIVE COMPOUNDS HYDROLYSIS et al., 2013). Such microscopic assays are often referred to as
LIVE/DEAD staining, although LIVE/DEAD is a trademark of
Fluorescein diacetate (30 ,60 -diacetyl-fluorescein) hydrolysis is Invitrogen, Thermo Fisher Scientific. LIVE/DEAD staining has
considered to be a simple and affordable method of estimating been benchmarked only for proteobacterial pathogen (mostly
microbial activity in various samples, including soils, sludges, E. coli) and has been reported to be prone to artifacts, including
marine sediments, and cell cultures (Fontvieille et al., 1992; incomplete stain penetration or false staining of live cells as
Yuan et al., 2007; Sánchez-Monedero et al., 2008; Gómez dead (Stewart and Franklin, 2008) in many cases. Similarly, FDA
et al., 2015; Mahu et al., 2018). In this assay, FDA, which is hydrolysis and its accumulation in active cells is often used in
colorless, is hydrolyzed by nonspecific esterases, proteases, combination with fluorescence-activated cell sorting (FACS) or
and lipases into green-colored fluorescein (Figure 6). The fluorescence microscopy to assess the number of stained cells
enzyme performing such hydrolysis can be free or membrane- and their relative fluorescence (Hoefel et al., 2003). FACS and
bound (Fontvieille et al., 1992; Adam and Duncan, 2001). As LIVE/DEAD assays will not be further discussed here, and focus
with tetrazolium, there are several FDA-based compounds, will remain on bulk assays.
such as 5(6)-carboxyfluorescein diacetate (carboxy-FDA) To measure metabolic rates in environmental samples or,
and 20 ,70 -dichlorofluorescein diacetate (chloromethyl-FDA), more rarely, in cultures, FDA hydrolysis is initiated by adding
leading to the formation of different hydrolyzed products, the FDA in a buffer (usually 60 mM phosphate buffer) to
in this case, 5-carboxyfluorescein (carboxyfluorescein) and the sample. After a determined incubation period, the reaction
20 ,70 -dichlorofluorescein (chloromethyl-fluorescein). However, is stopped using rather large amounts of organic solvents
[chloroform:methanol (2:1) or acetone]. In many environmental that the focus is put on terminal electron acceptors and that
samples, the solvent mixture also serves as a fluorescein NAD(P)+ /NAD(P)H have been discussed previously in the text.
extractant by dissolving membranes (Adam and Duncan, 2001;
Gaspar et al., 2001; Patle et al., 2018). In cell cultures, the
same methodology can be applied, or Triton X-100 can be Oxygen
used to permeabilize yeast cells, for example (Breeuwer et al., Oxygen is among the most used proxies to assess the metabolic
1995). After extraction, the fluorescein amount is quantified by activity of various organisms. This is also true for microorganisms
measuring either fluorescence or absorbance. Although FDA and microbial oxygen consumption rates, and their measurement
and its derivatives are mostly used for viability assays, their could be the topic of a review by itself. As a result, many
use as proxies to measure microbial metabolism remains very methods have been developed to assess oxygen consumption
interesting, as the molar absorption coefficient of fluorescein dye by heterotrophs or production by photosynthetic microbes
(E490 nm) lies between 67,000 and 79,000 cm−1 ·M−1 (Mota et al., (see review in Renger and Hanssum, 2009). Indeed, oxygen
1991). Such a high absorption coefficient compared to formazan concentration can be measured in solution using electrodes
(see above and Table 1) and its lower toxicity make it interesting (electrochemical sensors), optodes (optical-based sensors), or
for environmental studies. In addition, fluorometric detection is chemical assays such as the Winkler titration or directly
even more sensitive and can be used as well. However, the good in the headspace of sealed vials using laser spectrometry
detection of fluorescein is slightly compensated for by the lower (Bondyale-Juez et al., 2017).
water solubility of FDA. Indeed, many protocols recommend With respect to dissolved oxygen, the Winkler titration
diluting FDA in an organic solvent such as acetone, chloroform, (Winkler, 1888; Katznelson, 2004) was considered to be the most
dimethyl sulfoxide (DMSO), ethanol, or methanol where the accurate method for a long time (Bittig et al., 2018); however,
solubility is about 25 mg/ml. the method is demanding and rather time-consuming, as the
reaction of dissolved oxygen with manganese is rather slow
and can take up to 30 min. Therefore, the Winkler titration
ELECTRON ACCEPTOR CONSUMPTION is difficult to apply serially to assess the rapid consumption of
RATE oxygen. As a result, optical and electrochemical sensors have
been preferred in many cases (Kemker, 2014; Bondyale-Juez
Electron acceptors are key players in microbial metabolism, et al., 2017), especially as some systems have been introduced
providing energy for many other processes. Indeed, many as 96-well plates (Guarino et al., 2004; Dike et al., 2005).
microorganisms obtain their energy from redox reactions. Nowadays, both optical- and electrochemical-based sensors have
Among these redox reactions, aerobic respiration, anaerobic very good performance and can sense dissolved oxygen in an
respiration, and oxidation of reduced inorganic compounds rely aqueous system easily and accurately, usually with an error
on redox couples such as O2 /H2 O, NO3 − /NO2 − and Fe3+ /Fe2+ , below 1% (Briand et al., 2004; Tengberg et al., 2006; Renger and
for example (Konhauser, 2007; Oren, 2008). The consumption Hanssum, 2009). In addition, both types of electrodes can be
rate of the electron acceptor or the production rate of its reduced built as macro or micro types of sensors (Revsbech et al., 1983;
form is therefore a very good proxy to follow or estimate Visscher et al., 1991; Glud et al., 1999, 2005). Still, it is worth
microbial metabolism. As a result, many assays and techniques reviewing the minor differences between these types of sensors.
have been used for this purpose. Again, many of these assays Clark-type electrodes have been shown to require frequent
were intended for use with pure culture, and their use for calibration, to consume some oxygen, and to be sensitive to
environmental samples should be carefully evaluated, as there environmental factors (especially salinity, flow, temperature, and
could be many limitations or artifacts. The next sections will pressure). On the other hand, oxygen optodes do not consume
provide insights on some of the commonly used assays; however, oxygen and the signal is minimally affected by flow velocity
such a list cannot be considered as exhaustive. In particular, note and other environmental factors, except temperature. However,
in many commercial optodes, the temperature dependence is ways. Although nitrate (NO3 − ) and nitrite (NO2 − ) can be
automatically corrected by an attached thermistor2 . In the end, measured by high-performance liquid chromatography (HPLC),
the most important difference between optodes and electrode ion chromatography (IC), or ion-specific electrodes, the Griess
is the possibility to obtain 2D spatially resolved information by reaction (Griess, 1879) remains very popular to assess the
building planar optodes (Glud et al., 2005; Staal et al., 2011; potential for nitrate reduction and, if measured serially,
Tschiersch et al., 2011; Farhat et al., 2015), as microelectrodes denitrification rates. The original Griess reaction assesses the
(including needle optodes) are limited to 1D depth profiles amount of nitrite in the sample in two steps. First, nitrite reacts
or time series (Kuhl, 2005; Cotter et al., 2009; Riedel et al., under acidic conditions with sulfanilic acid, thus producing a
2013). Finally, a few commercial high-throughput products have diazonium cation. Second, the diazonium cation reacts with 1-
been released using optodes (or optode-like technology) and naphthylamine to produce a dark red water-soluble azo dye
allowing 96- or 384-well-plate format assays. Among them, the (Tsikas, 2007) (Figure 7). The Griess reaction is specific for nitrite
BD Oxygen Biosensor System is an oxygen-sensing microplate but can determine nitrate as well if it is properly reduced to nitrite
using silicone and an embedded fluorophore [tris 4,7-diphenyl- chemically or enzymatically. Such reduction is usually performed
1,10-phenanthroline ruthenium (II) chloride] at the bottom of by adding zinc powder to the sample solution. However,
the wells. The instrument has been used successfully to monitor alternative reduction methods using cadmium, vanadium, silver,
the growth and oxygen uptake rates of mammalian cell cultures or nitrate reductase have been considered as well (Sun et al.,
and bacterial cultures (Stitt et al., 2002; Guarino et al., 2004). 2003; Beda and Nedospasov, 2005; García-Robledo et al., 2014;
A similar system, the Agilent Seahorse XF Analyzer, offers more Wang S. et al., 2016).
analytical capacities but is intended for work with adherent Like many assays, the Griess reaction has been modified, and
cells (Gerencser et al., 2009), and as a result, very little data the original sulfanilic acid and 1-naphthylamine were replaced
have been obtained using this system on bacteria. Still, parasite by sulfanilamide and N-(1-naphthyl)ethylenediamine (NED or
investigations have been performed successfully (Shah-Simpson NEDA). Most variations of the Griess assay were reviewed
et al., 2016; Gonzalez-Ortiz et al., 2019). in Tsikas (2007).
Finally, although the measurement is a bit more indirect Finally, it must be noted that the low cost and simplicity
compared to dissolved oxygen measurement, oxygen can also of the Griess reaction have attracted the attention of a wide
be assessed in the headspace of sealed gas-tight vials using spectrum of microbiologists ranging from soil scientists to
laser spectroscopy. Indeed, tunable diode laser absorption clinical microbiologists.
spectroscopy (TDLAS) can be used to measure several gases
in gas phase. TDLAS measures the absorption of laser light in
the near- to mid-infrared wavelength. Detection of gases can Sulfate and Sulfide
be improved using techniques such as wavelength modulation For sulfate-reducing and sulfide-oxidizing bacteria, sulfates
spectroscopy (WMS) or frequency modulation spectroscopy and sulfides can be assessed by using HPLC or IC. In addition
(FMS) (Krishna and O’Byrne, 2016). It works well for many to these methods, which are used a lot, there are also simple
gases, including oxygen and carbon dioxide. TDLAS readings assays. For sulfate, barium sulfate precipitation is a very simple
are fast; however, current instruments have only one measuring assay that uses the low solubility of this salt [solubility at
channel and samples need to be read over a time series manually. 20◦ C = 2.42·10−3 g·L−1 − Ksp = 1.0842·10−10 (25◦ C)],
Still, some semiautomated systems are currently available for which is measured turbidimetrically (Coleman et al., 1972;
industrial applications, especially in the pharmaceutical industry US EPA, 1986; Kolmert et al., 2000). As a consequence, the
(Brückner et al., 2019). In addition, such instruments are under precipitation is often carried out using centrifuged or
a constant flow of nitrogen and therefore require a fair amount filtered samples. Moreover, to ensure similar precipitation
of that gas. TDLAS has been used in microbiology recently for conditions (and uniform crystal size distribution to improve
the detection of bacterial growth in pharmacological samples turbidimetric measurement reproducibility) between samples,
and clinical samples (such as blood cultures) based on O2 a conditioning reagent containing sodium chloride, ethanol,
and CO2 concentrations in vials (Brueckner et al., 2016, 2017; glycerol, and hydrochloric acid is used (its composition varies
Duncan et al., 2016; Shao et al., 2016, 2018). Furthermore, it is between authors and between available commercial assay
possible to detect bacterial growth linked to cystic fibrosis or the kits). Many authors have reported that the assay is rather
presence of Helicobacter pylori infection directly from exhaled air long and not appropriate for high-throughput measurement.
(Henderson et al., 2018) using other volatile compounds. Still, for slow-growing sulfate-reducing bacteria (SRB), the
assay allows serial measurements and determination of the
Nitrates (No3 − ) and Nitrites (No2 − ) sulfate reduction rate.
Because the energy yield of nitrate reduction is still rather In the case of bacteria reducing sulfide to elemental sulfur,
high compared to other types of anaerobic metabolism, it sulfides are more practical to measure, as many methods
is a commonly encountered type of metabolism that is also have been developed and can be applied to biological samples
found in pathogenic microbes. As a result, nitrate reduction (see review in Lawrence et al., 2000). In particular, hydrogen
is often studied in microbiology and assessed in several sulfide (HS− ) ion-sensitive electrodes are very practical and
are often used because they are available in macro and
2
https://www.unisense.com/MicroOptode micro format. With microelectrodes that are used in situ (or
ex situ), measurements are taken directly and possibly on- many of these metals (Gupta et al., 2011), the commercial
site. They have been extensively used in sediment, microbial versions of these electrodes are limited to a few of the
mat, and biofilm studies (Ito et al., 2002; Farías et al., metals (Cu, Hg, Ag, Cd). Therefore, atomic absorption or
2014; Pages et al., 2014; Wong et al., 2015). However, for MS is often used to measure their concentration. As these
macroelectrodes, especially if measurement is delayed until methods are not always available in a microbiology lab
the return to the lab, transportation and potential aeration and because some of the samples cannot be stored for a
might bias the results, as sulfides will oxidize rapidly. To long period, reduction rates are rarely measured with this
prevent such oxidation, commercial sulfide antioxidant buffer type of metabolism. Still, nonspecific assays (such as those
(SAOB) or 10 M NaOH can be used. Solutions such as zinc described above, FDA, or calorimetry; see below) can be useful
acetate should be avoided, as ion-selective electrodes only in such contexts.
measure free sulfides. Only iron (i.e., Fe2+ and Fe3+ ) can be assessed easily
On the other hand, sulfide assays relying on the formation using assays relying on the reaction of a chromogene with
of methylene blue have been very popular in microbiology iron II (phenanthroline, bathophenanthroline, ferrozine, and
(Reese et al., 2011). These assays rely on the reaction of ferrene) (Saywell and Cunningham, 1937; Goodwin and
dimethyl-paraphenylene diamine salts with sulfide to form Murphy, 1966; Hirayama and Nagasawa, 2017; Hopwood et al.,
methylene blue under acidic conditions. Absorbance of 2017; Hach, 2020). Many of these assays are available as
the methylene blue formed can be measured at 663 nm commercial kits and often allow the measurement of Fe2+
(E663 nm = 95,000 cm−1 ·M−1 ; Cenens and Schoonheydt, and total Fe by adding a reducing agent (ascorbic acid or
1988). To prevent oxidation of the sulfide, the samples can ammonium hydroxide) to convert Fe3+ into Fe2+ . Many
be fixed as ZnS using zinc acetate solution. The ZnS formed commercial kits do not provide the composition or the
is usually stable for 1 month. Upon acidification, ZnS can be type of assay present in the kit. However, it is possible
redissolved and assessed as described above. Many modifications to decipher them according to the measurement wavelength
of the assay have been used over time (see review in Lawrence of the assay (phenothroline and bathophenanthroline: 533–
et al., 2000; Reese et al., 2011). However, in microbiology and 535 nm; ferrozine: 563 nm; ferrene: 593 nm; Hirayama
the study of microbial mats, the modification of Pachmayr and Nagasawa, 2017). In some assays, this can even be
in 1960 (Pachmayr, 1960) using dimethyl-paraphenylene performed serially, making the assays very convenient. Indeed,
diamine sulfate (DPDS) is one of the most commonly used some commercial kits, such as the HACH test kit (LCK
(Trüper and Schlegel, 1964; Gallagher et al., 2012). Although 320), have all the reagents contained in one single assay
such assay is more work-intensive compared to ion-selective tube with breakable septa. In other assays, the determination
electrode (ISE) readings, the fact that the sample can be of Fe2+ and total Fe must be performed in parallel. The
fixed and processed at a later time point (up to 1 month) uses, advantages, and drawbacks of these assays have been
makes it useful in practice, as large batches of samples can reviewed by a geomicrobiologist (Braunschweig et al., 2012).
be processed at the same time. Furthermore, with respect to Usually, organisms that grow using iron redox reactions are
environmental studies, the assay is sensitive to metastable forms rather slow, and these assays are rapid and practical enough
of iron sulfide deposits [such as amorphous FeS nanoparticles, to be performed serially to determine iron oxidation or
Mackinawite (FeS), Greigite (Fe3 S4 ), or nanoparticle of pyrite reduction rates.
(FeS2 )] that might be present on cell surfaces or attached
to biofilms/extracellular polymeric substance (EPS) matrix
(Morse and Rickard, 2004; Rickard and Morse, 2005). In CARBON SOURCE CONSUMPTION AND
human microbiology, the assay has also been adapted for sulfate BY-PRODUCT RELEASE RATES
reduction occurring in the oral cavity responsible for oral
malodor (Kanehira et al., 2012). Carbon Sources
Carbon sources are the key to microbial metabolism, providing
Metal and Metalloid Ions carbon for biosynthetic processes but also for energy metabolism
Many metal ions can be biologically reduced (and less frequently in many cases. Current technologies (such as Biolog EcoPlate)
oxidized), thus releasing sufficient amounts of energy to using well-plate readers allow rapid screening of several
sustain growth processes. Among those metals, Fe, Mn, V, important carbon sources for their utilization and eventually
Cr, Cu, Mo, As, Hg, Se, Au, U, and Tc have been reported link such data with metagenomics data obtained separately
to be reduced by microorganisms (Lovley, 1993; Konhauser, (Lyons and Dobbs, 2012; Uroz et al., 2013; Gryta et al.,
2007; Oren, 2008). Among them, iron, which is the fourth 2014; Feigl et al., 2017). However, such an approach does
most abundant element in the Earth’s crust, has received not provide information on metabolic rates. Indeed, the rate
special attention, as Fe(II) can function as an electron source of carbon source consumption is directly linked to metabolic
and Fe(III) as a terminal electron acceptor under anoxic activity. In this context, it must be noted that metabolic
conditions for iron-reducing microorganisms. In this context, activity is not always related to growth, as microbes are
iron redox reactions have the potential to support substantial known to engage in futile cycles for several reasons (Neijssel
microbial populations in soil and sedimentary environments. et al., 1990; Russell and Cook, 1995; Qian and Beard,
Although ion-selective electrodes have been developed for 2006). Therefore, it is important to avoid making direct
links between growth, metabolic activity, and potential carbon enzymatic assays are very specific and provide more accurate
source utilization. One has to discriminate clearly between measurements. Two enzymatic assays exist to assess glucose
metabolic fingerprinting and metabolism measurement. Still, in various samples (Figure 9). The first assay is based on
a screening of the important carbon sources using EcoPlates glucose oxidation by glucose oxidase (EC: 1.1.3.4), which
might allow deciphering which assays must be performed generates H2 O2 and D-gluconic acid. After this step, hydrogen
at later stages. peroxide reacts with o-dianisidine in the presence of peroxidase
The variety of carbon sources that can be used by microbes (EC: 1.11.1.7) to form a colored compound in the reaction
is indeed matched by a high number of assays to assess solution. Finally, oxidized o-dianisidine reacts with sulfuric
them. Especially, there are many enzymatic assays matching the acid to form a more stable colored product (Sigma Aldrich).
substrates to be assessed. There are also plenty of chemical assays The other enzymatic assay used for glucose determination
that are useful for assessing the uses of various carbon sources. relies on hexokinase (EC: 2.7.1.1). Glucose is phosphorylated
by hexokinase, and the resulting glucose-6-phosphate is then
Carbohydrates oxidized to 6-phospho-gluconolactone by glucose-6-phosphate
Carbohydrates represent a large fraction of carbon sources dehydrogenase (EC: 1.1.1.49). The NADH produced results in
investigated in clinical or natural environments and added increased absorbance at 340 nm. Of note, glucose-6-phosphate
to solid and liquid culture media. As a consequence, many dehydrogenase usually uses NADP+ as a cofactor, but this
chemical assays have been developed to assess the amount of enzyme is rather unspecific with regard to the cofactor (NADP+
carbohydrates in such samples. All of these assays rely on or NAD+ ). Thus, in many commercial assays, NAD+ is used
the same general principle: carbohydrates (including bound because of its lower cost and better stability (Bondar and
carbohydrate and glycoprotein) are hydrolyzed using a strong Mead, 1974; Fuentealba et al., 2016). Both enzymatic assays
acid and heat. The reaction mixture contains a developing reagent are commercially available and cost roughly the same. Several
(phenols, orcinol, o-toluidine, anthrone, carbazole) that allows studies focused on glucose consumption used diabetic glucose
the development of a color measurable by spectrophotomeric meters [electrochemical sensors also based on glucose oxidase
means (Georges, 1971; de Toledo et al., 2012). Among these (sensing H2 O2 )] to measure glucose. The approach is cheap,
assays, the phenol–sulfuric acid assay from Dubois et al. (1951, although calibration is required as those meters have been
1956) (Figure 8) and the anthrone assay (Dreywood, 1946; optimized for blood tests and might have an offset in other
Morse, 1947; Trevelyan et al., 1952) have been the most media or solutions (Flavigny, 2014). Glucose-doped samples
popular for years and are used extensively in many labs. Both might also be of use for low-glucose concentrations or when
assays are inexpensive and work well for monosaccharides, validation is needed (authors’ personal observations). At this
polysaccharides, and complex polysaccharides such as EPS point, many attempts are made to “hack” such meters and
(Scott and Melvin, 1953; Dubois et al., 1956; Braissant et al., use them as bacterial contamination detection tools/platforms
2009). However, the anthrone assay works better for solutions (Flavigny, 2014; Wang et al., 2015). Finally, it must be noted
containing a single type of hexose because even sugars with that glucose sensors are among the most investigated types of
similar structures result in different rates and quantities of sensors. A recent study showed that over 9,000 publications
color development (Cui and Brummer, 2005). With respect to existed on the topic in Web of Science, and around 400
throughput, the original phenol–sulfuric acid assay is difficult to more appear every year (Oliver et al., 2009; Chen et al.,
adapt to microplate format (due to the high temperature reached 2013). As a consequence, glucose detection will still change
during the assay, incompatible with polystyrene well plates); quite a lot in the coming years and other assays are likely to
however, both that assay and the anthrone assay have been become available.
modified extensively and optimized for well plates (Laurentin
and Edwards, 2003; Leyva et al., 2008). Often, those modified Organic Acids
methods involve some incubation at higher temperature (90◦ C Among the potential carbon sources used by microbes or
or above) to speed up the reaction. Many commercial versions the by-products released by their metabolism, organic acids
of such chemical assays are sold as “carbohydrate assay” or have received a lot of attention due to their role in food
“total carbohydrate assay,” making them readily available. For production, plant growth, diseases, and weathering processes.
these assays, samples can be collected serially and frozen. At The measurement of organic acids in various samples is
the end of the experimental measurement, the assay can be often performed using IC or HPLC (Takao, 1965; Adams
performed easily and the well plate format allows rather high et al., 1984; Trifirò et al., 1997; Lefebvre et al., 2002; Tsangalis
throughput. Therefore, carbohydrate consumption rates can be and Shah, 2004; Klinke et al., 2009; Wojtczak et al., 2010)
easily determined during an in vitro assay where an excess of one as well as capillary electrophoresis (Kudrjashova et al.,
sugar is used in the medium. 2004). However, chromatography and electrophoresis often
demand some preparation, during which sample recovery
Glucose might not be optimal. For single organic acids, many
Among carbohydrates, glucose is probably the most used and commercial enzymatic assays can be used to detect the
monitored, and it can be easily assessed using the assays most commonly encountered ones (Table 2). These assays
described above. However, for samples containing other sugars or often rely on the production or consumption of NADH
interfering substances (toluene, for example; Devor et al., 1964), directly detected by a change in absorbance at 340 nm.
TABLE 2 | Non-exhaustive list of organic acid assays and their enzymatic and detection systems.
Assays from providers that do not release the enzyme systems used or the nature of the redox or fluorimetric probe used for final detection were not included. Data
compiled from the websites of the following providers: Megazyme, Abcam, Cell Biolabs Inc., Trinity Biotech, Libios, and Sciencell.
AB, alamarBlue (resazurin); WST, tetrazolium-based dye; MBTH, 3-methyl-2-benzothi-azolinone hydrozone; DMAB, 3-(dimethylamino)benzoic acid. **First standard
solution used for calibration in manufacturer booklet.
NA, data not available.
INCORPORATION ASSAYS (TO DW) > lipids (8%–9% DW) > DNA (2%–3% DW) (Taymaz-
MONITOR MICROBIAL ACTIVITY) Nikerel et al., 2010; Liao et al., 2011). This makes the choice of
the target crucial in order to have the best possible monitoring of
The incorporation of labeled or analog substrates in microbial the incorporation, depending on the extraction and recovery of
biomass has been widely used (and would certainly deserve the target molecule to be labeled from the sample (or culture).
a review on its own). The incorporation rate of the selected Furthermore, one has to keep in mind that the proportions
compounds is assumed to be proportional to the metabolic of these macromolecules might vary a lot depending on the
activity and the viability of anabolically active microbes. growth conditions and growth rate (Taymaz-Nikerel et al., 2010;
As a result, many incorporation assays relying on different Liao et al., 2011).
detection methodologies have evolved. In addition, many Overall, the compounds used for incorporation assays fall
of these techniques are compatible with further use of under three main categories: radioisotopes, stable isotopes, and
microscopy and single-cell analysis as well as (meta)genomics substrate analogs (Table 3).
and (meta)proteomics tools (not discussed within the scope of
this review; see Hatzenpichler et al., 2020, for more information), Radioisotope Incorporation
thus making such incorporation assay very attractive when Radioisotopes are easily detected because of the ionizing
further functional or taxonomic characterization of a system radiations they emit. The most commonly used radiolabeled
is considered. The sensitivity of incorporation assays depends compounds are 3H-thymidine and 3H-leucine or 14C-leucine.
on the detection technology on one end, but also the relative These radiolabeled substrates have been used separately or in
amounts of cell component labeled on the other end. In combination (3H-thymidine and 14C-leucine). Thymidine is
microbial cells, the relative abundance of macromolecules is as incorporated into microbial DNA and thus provides a measure
follows: proteins [52%–70% dry weight (DW)] > RNA (4%–20% of DNA synthesis. Similarly, leucine is incorporated into proteins
Radioactive
3H-thymidine DNA Scintillation counter or microautoradiography (MAR)
3H-leucine Protein Scintillation counter or microautoradiography (MAR)
14C-leucine Protein Scintillation counter or microautoradiography (MAR)
14C-acetate All Scintillation counter or microautoradiography (MAR)
14C-glucose All Scintillation counter or microautoradiography (MAR)
3H-hypoxanthine All Scintillation counter or microautoradiography (MAR)
Stable isotopes
13C All Raman, mass spectrometry, density separation
15N Protein DNA Raman, mass spectrometry, density separation
2H nucleoside DNA Raman, mass spectrometry, density separation
2H (2H2O) Lipids Raman, mass spectrometry, density separation
Substrate analogs
L -Azidohomoalanine (AHA) Protein (methionine analog) Click chemistry with matching fluorescent or affinity tag
L -Homopropargylglycine (HPG) Protein (methionine analog) Click chemistry with matching fluorescent or affinity tag
Ethynyl-D-alanine (EDA) Protein (alanine analog) Click chemistry with matching fluorescent or affinity tag
Azido-D-alanine (ADA) Protein (alanine analog) Click chemistry with matching fluorescent or affinity tag
O-Propargyl-puromycin (OPP) Protein (alanine analog) Click chemistry with matching fluorescent or affinity tag
N-Azidoacetylmannosamine-tetraacylated (Ac4ManNAz) Glycosylated protein Click chemistry with matching fluorescent or affinity tag
N-Azidoacetylglucosamine-tetraacylated (Ac4GlcNAz) Glycosylated protein Click chemistry with matching fluorescent or affinity tag
N-Azidoacetylgalactosamine-tetraacylated (Ac4GalNAz) Glycosylated protein Click chemistry with matching fluorescent or affinity tag
5-Bromo-20 -deoxyuridine (BrdU) DNA (thymidine analog) Immunostaining or immunocapture
5-Ethynyl-20 -deoxyuridine (EDU) DNA (thymidine analog) Click chemistry with matching fluorescent or affinity tag
(20 S)-20 -deoxy-20 -fluoro-5-ethynyluridine (F-ara-EdU) DNA (thymidine analog) Click chemistry with matching fluorescent or affinity tag
5-Ethynyl uridine (EU) RNA (uridine analog) Click chemistry with matching fluorescent or affinity tag
Alkynyl palmitic acid Lipids Click chemistry with matching fluorescent or affinity tag
Azido palmitic acid Lipids Click chemistry with matching fluorescent or affinity tag
Alkynyl myristic acid Lipids Click chemistry with matching fluorescent or affinity tag
Azido myristic acid Lipids Click chemistry with matching fluorescent or affinity tag
Alkynyl stearic acid Lipids Click chemistry with matching fluorescent or affinity tag
Azido stearic acid Lipids Click chemistry with matching fluorescent or affinity tag
and acts as a proxy for protein synthesis. The combination of several indicators gathered from MS data have been recognized as
both allows the measurements of protein synthesis and DNA useful proxies for metabolic rates and carbon fluxes. In particular,
synthesis in a single experiment. This approach assumes that labeling ratio (lr), relative isotope abundance (RIA), and the
thymidine and/or leucine is taken up into the cells; however, some shape of the isotope pattern of specific peptides can provide
microbes, especially strict autotrophs and oligotrophs, lack the valuable information (Jehmlich et al., 2010; Taubert et al., 2011;
necessary transporters for the uptake of many organic molecules, Von Bergen et al., 2013). The lr is the ratio of the intensity of
including amino acids and nucleotides (Pérez et al., 2010). the isotope pattern of labeled peptide and the total intensity of
Practically, samples are incubated with radiolabeled substrates isotope patterns of unlabeled and labeled peptides. This ratio is
at a final concentration within the µM range (from 1 µm for considered to be an indicator of protein synthesis and turnover in
thymidine to 15 µM for leucine; see an example in Tuominen, time-resolved analysis. Indeed, the evolution of the lr over time
1995), ensuring saturation (Bååth, 1998; Bloem and Bolhuis, closely matches the growth curve for protein expressed under
2009). It must be noted that such concentrations are at least growth conditions (Taubert et al., 2011; Von Bergen et al., 2013)
two to four orders of magnitude higher than the concentrations and can be processed using growth curve fitting equations. For
of these compounds in the environment, where they typically such analysis, the choice of the protein or peptides analyzed is
do not exceed the nM range (Jørgensen, 1982, 1987). This therefore crucial, and several peptides might be analyzed. RIA
certainly has an effect and must be considered as a potential is the percentage of stable isotope incorporation estimated using
limitation of such approach. The incubation is usually short to the extent of mass shift of the peptide peak. It is a proxy for the
avoid inducing changes in the microbial community structure metabolization of carbon (or nitrogen) (Von Bergen et al., 2013).
and potential deleterious effects of ionizing radiation and ranges Finally, an ideal Gaussian distribution of the m/z (mass over
from a few minutes (Tuominen, 1995) to a few hours (Bååth, charge) values of a peptide indicates direct substrate utilization.
1998; Bloem and Bolhuis, 2009) depending on the nature and On the other hand, m/z value distribution for a peptide showing
activity of the sample. After incubation, the incorporation is an isotope pattern with negative skewness (i.e., differing from
stopped by cooling and/or adding ethanol or trichloroacetic the ideal Gaussian distribution and tailing toward lower m/z
acid (TCA). Labeled protein or DNA is then extracted and values) indicates indirect 13C substrate metabolization (e.g.,
precipitated, taking care to remove unincorporated radiolabeled through cross-feeding) (Von Bergen et al., 2013). These three
thymidine and leucine. Finally, after a final solubilization step, indicators have been investigated with protein-SIP until now;
labeled macromolecules are determined using a scintillation however, one can assume that they can be used with PLFA-
counter and the incorporation rate can be calculated. The SIP as well.
use of radiolabeled isotopes is becoming less frequent as As an alternative to MS, Raman microspectroscopy can
less demanding alternatives (in terms of labor, safety, and be used in combination with optical tweezers or single-cell
waste management) are developed (see following sections). ejection to sort and separate labeled from unlabeled cells with
Still, other radioactive substrates are still in use for specific high accuracy. The incorporation of heavy isotopes in the
purposes such as in parasitology (3H hypoxanthine) and cell macromolecules results in a shift of the Raman peaks
environmental science (14C acetate, 14C glucose) in combination toward lower wavenumbers compared with unlabeled cell spectra
with microautoradiography (MAR) (Brun and Kunz, 1989; Lee (Von Bergen et al., 2013; Wang et al., 2013; Jing et al.,
et al., 1999; Chidthaisong and Conrad, 2000; Nielsen et al., 2003a; 2018). That detectable Raman shift can be used to manually
Maerki et al., 2006). or automatically trigger the use of optical tweezers or single-
cell ejection system to allow rapid screening of cells and
Stable Isotope Probing keep them for further analysis (metagenomics) and eventual
Stable isotope probing (SIP) refers to the use of stable isotope- metabolic profiling (Von Bergen et al., 2013; Wang et al., 2013;
labeled substrates to probe the microbial utilization of these Jing et al., 2018; Lee et al., 2019). In this context, Raman
specific substrates to build their macromolecules (Jehmlich et al., spectra can be used directly to monitor the incorporation rate,
2010; Emerson et al., 2017; Hatzenpichler et al., 2020). As a result, as the shift observed is proportional to the incorporation of
DNA-, RNA-, phospholipid fatty acid (PLFA)-, and protein-SIP heavy isotopes and can be from the bulk biomass (Li et al.,
are possible using different stable isotopes (mostly 2H, 13C, 2013) down to the single-cell level using spontaneous Raman
15N, and 18O). Comparing the different SIP targets, one can microspectroscopy (Wang et al., 2013; Jing et al., 2018; Lee
see a trade-off between the amount of incorporation required, et al., 2019) or surface-enhanced Raman spectroscopy (SERS)
the workload, and the taxonomic information gained (Jehmlich (Wang Y. et al., 2016).
et al., 2010). Indeed, protein-SIP can provide some taxonomic
information; however, the gold standard remains DNA-SIP. In Substrate Analog Probing
most studies, the use of SIP is intended to gather taxonomic or Substrate analog probing is a term introduced by Hatzenpichler
proteomic information about the anabolically active part of the (Hatzenpichler et al., 2020) to differentiate the approach
microbial population. Much valuable information on metabolic from SIP. This approach uses synthetic compounds that are
rates can be gathered in the analytical process as well. Most structurally and/or functionally analogous to natural molecules.
SIP studies rely on the extraction of target macromolecules (i.e., Such analogs are incorporated into cell macromolecules due
DNA, RNA, phospholipids, and protein) and further analysis to enzyme promiscuity. Some of these substrate analogs
using liquid chromatography coupled with MS. In this context, already bear a fluorescent tag, allowing their direct detection;
FIGURE 7 | Scheme showing reactions involved in determination of nitrite and nitrate. Some reducing agents to convert nitrate to nitrite are listed (see text for a
more complete list).
however, most analogs must be detected after incorporation Similarly to radiolabeled thymidine incorporation, 5-bromo-
using immunocapture, immunostaining, or “click chemistry” 20 -deoxyuridine (BrdU) has been used in the same way. Indeed,
(i.e., azide–alkyne cycloaddition; Liang and Astruc, 2011) BrdU is an analog of the DNA precursor thymidine. When
reactions with an appropriate fluorescent or affinity tag. it is added to a sample containing growing cells (culture or
The variety of substrate analogs available nowadays allows environmental sample), the cells incorporate BrdU into their
monitoring of the incorporation of substrate analogs into DNA instead of thymidine. The BrdU-containing DNA is
all types of macromolecules (i.e., DNA, RNA, protein, and then detected using an anti-BrdU antibody. Then, a secondary
lipids; Table 3). antibody specific for the anti-BrdU primary antibody is used to
FIGURE 9 | Example of enzymatic assay for glucose using glucose oxidase (top panel) or hexokinase (bottom panel). Note that glucose-6-phosphate
dehydrogenase usually uses NADP+ as cofactor; however, this enzyme is rather unspecific with regard to the cofactor (NADP+ or NAD+ ). Thus, in many commercial
assays, NAD+ is used because of its lower cost and better stability (Bondar and Mead, 1974; Fuentealba et al., 2016).
capture the DNA (with the help of paramagnetic beads), thus Hatzenpichler et al., 2020). In BONCAT, a methionine analog
providing a new sample of reduced complexity that corresponds such as azide-bearing azidohomoalanine (AHA) or alkyne-
to cells effectively dividing (Men et al., 2011; Robbins et al., 2011), bearing homopropargylglycine (HPG; syn: 2-amino-5-hexynoic
which can be further analyzed using molecular methods (Robbins acid) is used to label newly formed proteins (Landgraf et al.,
et al., 2011). Alternatively, for visualization or quantification 2015). Indeed, methionine-tRNA ligase (EC: 6.1.1.10: syn
purposes, the secondary antibody can be coupled with a methionyl-tRNA synthetase) can accommodate amino acid
fluorescent tag. As for other incorporation assays, one can assume analogs such as homopropargylglycine (HPG) and AHA but
that the incorporation rate is proportional to the metabolic at a slower rate. Between these two methionine analogs, HPG
activity. However, it must be noted that in some biofilms or when incorporation is still slower than AHA incorporation (Kiick et al.,
experiencing limitation in nitrogen or phosphorus availability, 2002; Sherratt et al., 2017). After an incubation step (preferably
bacteria can show high metabolic activity but poor growth and in a medium without methionine), the cells are usually fixed to
thus very low DNA synthesis. In addition, it was shown that many increase sample stability during storage and then labeled using
species are not able to incorporate BrdU and that interpretation click chemistry (Figure 10) using the matching azide or alkyne
should be done with care when using BrdU. Urbach et al. (1999) tag. A variety of fluorescent tags have been developed and used
stated that “while BrdU incorporation can be used to prove in combinations with FACS and microscopy to detect and/or sort
that specific populations of bacteria in a natural ecosystem are anabolically active cells (Singer et al., 2017).
growing, this method cannot be used conversely to prove that a Similarly, biotin tags allowing quantification, affinity
population is not growing, unless it is also demonstrated that the purification, and further identification of protein using MS
species in question can assimilate BrdU.” have been used as well (Landgraf et al., 2015). Although
Among all the methods developed, bio-orthogonal BONCAT is intended more for further genomic or proteomic
noncanonical amino acid tagging (BONCAT) has garnered characterization, labeling rates of cells or specific proteins can
much interest because of its compatibility with many other also be used as a proxy for metabolic rates (Hatzenpichler
approaches such as FACS, fluorescence in situ hybridization et al., 2014). Indeed, the evolution of fluorescence of a culture
(FISH), nanoscale secondary ion mass spectrometry (nanoSIMS), or from extracted proteins matches 3H-leucine incorporation
(meta)genomics, and (meta)proteomics (Landgraf et al., 2015; (Leizeaga et al., 2017).
FIGURE 10 | Simplified sketch of bio-orthogonal noncanonical amino acid tagging (BONCAT) procedure using azido-alanine and the following click chemistry
reaction with matching Oregon Green 488 alkyne fluorescent probe. Note that for simplicity, the sketch only shows extracellular azidohomoalanine (AHA); however,
after the click reaction, both intracellular and extracellular AHA-labeled peptides are detected.
data that can be correlated or combined (Nocker et al., Moreover, calorimetry can provide an additional equation to
2011). Indeed, several studies have correlated two or more solve this system. Indeed, using the enthalpy of reaction provides
of the abovementioned assays for microbes (Cooney et al., an additional equation that can further help solve the equation
1969; Rodriguez et al., 2011; Braissant et al., 2015) or higher system. The enthalpy of reaction (1Hrxn ) measured by the
organisms (Braeckman et al., 2002). This is important, as it calorimeter can also be calculated using enthalpy of formation
allows rapid validation of the use of a technique or deciphering (1Hf ) or of combustion (1Hc ); using enthalpy of combustion
between different types of metabolism. In this context, a simplifies the equation even more (see details in Maskow and
lack of correlation might sometimes be more informative Harms, 2006; Barros et al., 2010; Maskow et al., 2010).
than correlation itself. For example, high activity measured 1Hrxn = −(1Hc Cbiomass + f·1Hc H − a·1Hc Cglc − c·1Hc
by an assay without matching electron acceptor reduction HNH4 ) (from Maskow et al., 2010)
might point to the use of an alternative electron acceptor The enthalpies of formation and combustion of organic
(NO3 − , for example) or fermentation (sensu stricto). Similarly, matter, and especially organic matter from microbial biomass,
calorespirometric ratios (heat per O2 and heat per CO2 ) can be found in several review papers (Cordier et al., 1987; von
can give valuable information. Heat per O2 can help detect Stockar et al., 1993; Popovic, 2019), thus making this approach
the presence of anaerobic processes, and heat per CO2 can easy to implement with relatively high accuracy.
provide hints on the type of carbon source used (carbohydrate, However, such an approach requires that the metabolism of
protein, or lipids) (Hansen et al., 2004). For heat per O2 , the organism (or group of organisms) is already known and that
the Thornton rule states that the enthalpy of combustion of the organisms have been characterized up to the point where the
organic compounds is roughly constant when expressed per growth equation can be written in an accurate manner.
mole of O2 . The average value of this ratio is −455 ± 15
kJ·mol−1 O2 ; that is, −107 to −120 kJ·mol−1 e- (Thornton,
1917; Hansen et al., 2004; Maskow et al., 2010; Von Stockar, FUTURE PERSPECTIVES
2013). Therefore, for respiring organisms (and irrespective of the
carbon source), the ratio can be used to estimate these parameters Many of these assays have been used for a long time and have been
(carbon source consumption or oxygen consumption). More improved and optimized using new chemicals and instruments.
importantly, deviation from the oxycaloric ratio can provide Still, as mentioned for glucose, many studies continue to improve
information on the presence of anaerobic processes and or adapt (not to say “hack” or “exploit”) these methods and
estimation of the fraction of anaerobic metabolism at steady approaches. Among the possible promising approaches for many
state. Similarly, heat per CO2 is a proxy for efficiency, as it of these assays, the lab on a chip is very appealing. Microfluidic
is an approximate measure of the rate of catabolism over the systems, piezoelectric pumps, and sub-millimeter size sensors
rates of catabolism plus anabolism. Low values of heat per make it possible to build an array of sensors that can monitor
CO2 indicate that only little energy is lost from catabolism different compounds over time and thus provide real-time
(Hansen et al., 2004; Wadsö and Hansen, 2015). Still, care must monitoring of metabolic rates. It is likely that, in the future,
be taken when using such ratios, as the presence or use of many of these assays (or replacements for some of them) will
nitrogen-containing compounds (such as urea) and peroxides be combined on single disposable chips. Alternatively, for now,
might lead to significant deviation from the Thornton rule 3D printing and rapid prototyping methods also allow rapid
(Hansen et al., 2004; Wadsö and Hansen, 2015). In addition, development of microfluidic devices that can incorporate such
calorespirometric ratios overall for many types of metabolism, new sensors. Finally, it must be noted that such chips are
even more complex types combining several of these assays, can very close to becoming reality, as several authors have already
provide a rather complete metabolic budget (carbon, nitrogen, presented multiple parameters using separate microprobes or
oxygen, energy). An example for glucose respiration is given microfluidic chips simultaneously measuring pH, OD, and
below: dissolved oxygen, for example (Wong et al., 2015; Guimer et al.,
2019). Still, as this is a rapidly evolving field, many new sensor
types might appear in the next years.
aC6 H12 O6 + bO2 + cNH4 + → CH1.8 O0.5 N0.2 + dCO2
+ eH2 O + fH+ AUTHOR CONTRIBUTIONS
The following conservation equations can now be written: OB and MA-F drafted and wrote the manuscript. TW and GB
(note that for the calculations below, d, e, and f are negative) critically reviewed the manuscript and improved it. All authors
C conservation: 6a + 1 + d = 0 read and approved the submitted version.
H conservation: 12a + 4c + 1.8 + 2e + f = 0
O conservation: 6a + 2b + 0.5 + 2d + e = 0
N conservation: c + 0.2 = 0 ACKNOWLEDGMENTS
Charge conservation: c + f = 0
As these equations clearly show, all variables are not OB wishes to thank the Merian Iselin Stiftung for its support
independent, and solving for only a few of the parameters and the colleagues of the Merian Iselin Klinik for valuable
(a, b, c, d, e, and f ) allows for determining the other ones. discussions.
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