RNA Biomarker Trends across Type I and Type II Aerobic Methanotrophs in Response to Methane Oxidation Rates and Transcriptome Response to Short-Term Methane and Oxygen Limitation in Methylomicrobium album BG8

Abstract

Methanotrophs are naturally occurring microorganisms capable of oxidizing methane, having an impact on global net methane emissions. Additionally, they have also gained interest for their biotechnological applications in single-cell protein production, biofuels, and bioplastics.

Full text

RNA Biomarker Trends across Type I and Type II Aerobic
Methanotrophs in Response to Methane Oxidation Rates and
Transcriptome Response to Short-Term Methane and Oxygen
Limitation in Methylomicrobium album BG8
Egidio F. Tentori,
a
*Shania Fang,
a
Ruth E. Richardson
a
a
School of Civil and Environmental Engineering, Cornell University, Ithaca, New York, USA
ABSTRACT Methanotrophs, which help regulate atmospheric levels of methane, are
active in diverse natural and man-made environments. This range of habitats and the
feast-famine cycles seen by many environmental methanotrophs suggest that methano-
trophs dynamically mediate rates of methane oxidation. Global methane budgets require
ways to account for this variability in time and space. Functional gene biomarker tran-
scripts are increasingly studied to inform the dynamics of diverse biogeochemical cycles.
Previously, per-cell transcript levels of the methane oxidation biomarker pmoA were found
to vary quantitatively with respect to methane oxidation rates in the model aerobic meth-
anotroph Methylosinus trichosporium OB3b. In the present study, these trends were
explored for two additional aerobic methanotroph pure cultures grown in membrane bio-
reactors, Methylocystis parvus OBBP and Methylomicrobium album BG8. At steady-state
conditions, per-cell pmoA mRNA transcript levels strongly correlated with per-cell methane
oxidation across the three methanotrophs across many orders of magnitude of activity
(R
2
=0.91).TheinclusionofbothtypeIandtypeIIaerobicmethanotrophssuggestsa
universal trend between in situ activity level and pmoA RNA biomarker levels which can
aid in improving estimates of both subsurface and atmospheric methane. Additionally,
genome-wide expression data (obtained by transcriptome sequencing [RNA-seq]) were
used to explore transcriptomic responses of steady-state M. album BG8 cultures to short-
term CH
4
and O
2
limitation. These limitations induced regulation of genes involved in
central carbon metabolism (including carbon storage), cell motility, and stress response.
IMPORTANCE Methanotrophs are naturally occurring microorganisms capable of oxidizing
methane, having an impact on global net methane emissions. Additionally, they have
also gained interest for their biotechnological applications in single-cell protein produc-
tion, biofuels, and bioplastics. Having better ways of measuring methanotroph activity
and understanding how methanotrophs respond to changing conditions is imperative for
both optimization in controlled-growth applications and understanding in situ methane
oxidation rates. In this study, we explored the applicability of methane oxidation bio-
markers as a universal indicator of methanotrophic activity and explored methanotroph
transcriptomic response to short-term changes in substrate availability. Our results contrib-
ute to better understanding the activity of aerobic methanotrophs, their core metabolic
pathways, and their stress responses.
KEYWORDS methanotrophs, methane oxidation, Methylomicrobium album BG8,
Methylocystis parvus OBBP, RNA-seq, pmoA, biomarkers
Methanotrophs are microbes capable of getting all their carbon and energy from
methane, the second most abundant greenhouse gas after carbon dioxide (1, 2).
Methanotrophs represent the only known biological methane sink and play a pivotal
Editor Jeffrey A. Gralnick, University of
Minnesota
Copyright © 2022 Tentori et al. This is an
open-access article distributed under the terms
of the Creative Commons Attribution 4.0
International license.
Address correspondence to Egidio F. Tentori,
eft35@cornell.edu.
*Present address: Egidio F. Tentori, Gradient,
Boston, Massachusetts, USA.
The authors declare no conflict of interest.
Received 6 May 2022
Accepted 21 May 2022
Published 9 June 2022
May/June 2022 Volume 10 Issue 3 10.1128/spectrum.00003-22 1
RESEARCH ARTICLE

role in the global methane cycle (3). Methanotroph-based biotechnologies, coupling
methane oxidation with production of value-added products like methanol, biopoly-
mers, single-cell proteins, lipids, and enzymes, have been gaining interest (4–6).
Therefore, they represent a possible lynchpin in biorefinery scenarios for conversion of
methane and/or biogas to bioproducts.
Methanotrophic bacteria are found in the phyla Proteobacteria and, more recently,
Verrucomicrobia (7, 8) and NC10 (9). The most widely studied methanotrophs are from
the classes Gammaproteobacteria and Alphaproteobacteria, and based on physiological
characteristics, they were historically divided into type I and type II methanotrophs,
respectively (10, 11). However, methanotrophs can exhibit considerable metabolic flex-
ibility even among methanotrophs of the same type. For example, they are capable of
using nitrate or ammonium as a nitrogen source (12, 13), nitrogen fixation (14), carbon
accumulation under nutrient-limited conditions (15, 16), and long-term survival under
starvation conditions (17–21).
Adefining characteristic shared by almost all methanotrophic bacteria is methane
monooxygenase (MMO) enzymes, which initiate methane oxidation (22). Methane is
converted to methanol, followed by formaldehyde, which can be either incorporated
as biomass or ultimately converted to CO
2
. Two distinct MMOs can be found in metha-
notrophs, particulate methane monooxygenase (pMMO) and soluble methane mono-
oxygenase (sMMO). Methanotrophs that possess both MMOs express pMMO under
high copper-to-biomass ratios and sMMO at low copper-to-biomass ratios (23). Gene
and gene expression amounts of pMMO and sMMO subunits, genes pmoA and mmoX,
respectively, are used to quantify populations of methanotrophs, their activities, and
their transcriptional and phenotypic response to changes in conditions (24–26). As
nearly all aerobic methanotrophs possess pMMO, gene pmoA is the preferred bio-
marker to determine both their abundance and methane oxidation activity.
Approaches that quantify both biomarker mRNA transcripts and/or enzymes in
addition to microbial amounts have been suggested as estimators of microbial contri-
butions in biogeochemically relevant processes (27). The utility of protein and mRNA
biomarkers in microbial communities has been demonstrated previously, in both iden-
tifying microbial function and determining in situ activities of specific community
members (28–31). Periods of temporal variations affecting microbial activity would also
be reflected by changes in their respective mRNA or enzyme pools.
In methanotrophs, increases in pmoA gene copies and pmoA transcript levels correlate
with increased methane oxidation (6, 32–34) and have been proposed as quantitative indi-
cators of their activity (35, 36). Recently, a strong correlation between steady-state per-cell
pmoA transcript levels and per-cell methane oxidation rate was demonstrated in the type I
methanotroph Methylosinus trichosporium OB3b grown in membrane bioreactors (37). If
similar relationships between biomarker amounts and activity are shared across different
methanotrophs, they could allow dynamic inference of in situ methane oxidation rates.
The goal of this study was to explore correlations between methane oxidation bio-
marker amounts and methanotrophic activity across aerobic methanotrophs species and
obtain further insight into their transcriptomic response to dynamic conditions of substrate
availability. To assess this, aerobic methanotrophs Methylocystis parvus OBBP (type II) and
Methylomicrobium album BG8 (type I) were grown in membrane bioreactors under differ-
ent conditions where biomarker pmoA gene and transcript amounts (via quantitative PCR
[qPCR] and reverse transcription-qPCR) were measured. Additionally, effects of short-term
(less than one retention time [RT]) substrate (O
2
and CH
4
) limitation and recovery on RNA
biomarker expression were explored in M. album BG8 using a targeted qPCR approach and
RNA sequencing to determine temporal expression patterns under dynamic conditions
influenced by environmental factors.
RESULTS AND DISCUSSION
Steady-state reactor performance. Reactor flow rates were monitored, and reten-
tion times (RTs) were consistent throughout operation. Reactors had average RTs of
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2.8, 4.3, 5.7, and 9.1 days for M. album BG8 reactors and 2.8, 4.3, 5.8, and 9.0 days for M.
parvus OBBP (see Fig. S1 in the supplemental material). RTs ensured that the methano-
troph cultures in these reactors could reach distinct steady-state growth conditions.
Reactors were considered steady state when biomass, oxygen, and methane levels
were not changing significantly. All reactors reached steady-state conditions at all four
RTs (Fig. 1); duration, average biomass and substrate levels, and methane oxidation
rates for steady-state periods are provided in Tables S1 and S2.
Steady-state periods of M. album BG8 reactors are provided in Table S1 and shown in
Fig. 1A and B. Dissolved-methane concentrations ranged between 0.21 and 1.68 mg L
21
,
and oxygen concentrations ranged from 3.77 to 5.26 mg L
21
, decreasing with increasing
retention time. Steady-state periods of the M. parvus OBBP reactors are provided in Table S2
and shown in Fig. 1C and D. Dissolved-methane concentrations ranged between 0.42 and
2.16 mg L
21
, while oxygen concentrations varied only slightly across all RTs, ranging from
4.19 to 5.41 mg L
21
. Both cultures followed the trend of increasing biomass with increasing
retention time. Overall, biomass levels for M. album BG8 reactors were higher than for M.
parvus OBBP for reactors with equivalent retention times. Differences in biomass amounts
could be from differences in growth rate, internal carbon storage, and intracytoplasmic
membrane (ICM) amounts, which differ between type I and type II methanotrophs (38).
Methane levels observed during substrate limitation periods for both cultures were not
always below reported methanotroph half-saturation constant (K
S
) values reported in the lit-
erature, which range from 0.06 to 1.48 mg L
21
(39–41). However, the observed levels were
enough to affect methanotroph growth and activity. It is known that K
S
values are system
specific(e.g.,impactedsignificantly with stirring rate) and could be higher in the case of the
methanotrophs grown in the membrane bioreactors. Volume-normalized methane oxida-
tion rates were consistent across retention times and cultures (0.095 to 0.105 mg CH
4
mL
21
day
21
) (Tables S1 and S2). Biomass-normalized methane oxidation rates decreased
with increasing retention times for both cultures, reflecting the increase in biomass with
retention time. Reactors had similar overall methane consumption rates while having dis-
tinct biomass levels. Differences in biomass-normalized methane oxidation rates implied dif-
ferent activities of the reactor methanotroph populations. Methane oxidation rates in this
FIG 1 Performance of M. album BG8 and M. parvus OBBP reactors. (A) M. album BG8, 2.8-day RT, CH
4
limitation, transition to 5.7-day
RT; (B) M. album BG8, 4.3-day RT, O
2
limitation, transition to 9.1-day RT; (C) M. parvus OBBP, 2.8-day RT, 5.8-day RT; (D) M. parvus
OBBP, 4.3-day RT, 9.0-day RT. Aqueous concentrations of CH
4
and O
2
, left yaxis, and biomass, right yaxis. Data are means 6
standard deviations from triplicate reactors.
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study agreed with reported literature values of 0.00014 to 0.14 mg CH
4
mL
21
day
21
(42) and
0.48 to 3.85 mg CH
4
mg cell (dry weight)
21
day
21
(37, 43) for aerobic methanotrophs.
Biomarker amounts and oxidation rates under steady-state conditions. Correlations
between steady-state methane oxidation activity and cell and pmoA transcript amounts for
M. album BG8 and M. parvus OBBP reactors were determined for the sample times indi-
cated in Tables 1 and 2 and Fig. S2. For both methanotrophs, reactor methane oxidation
rates and pmoA transcript amounts were normalized by corresponding genome amounts
to obtain per-cell methane oxidation rates and per-cell pmoA transcript levels (Fig. 2).
The range of per-cell pmoA transcript levels across oxidation rates suggests a wide
range of activities for the methanotroph cultures in this study. M. album BG8 cultures
exhibited a wider range of both per-cell transcript amounts (0.0004 to 60.84) and per-
cell methane oxidation rates (0.005 to 1.681) than did M. parvus OBBP, 0.0002 to 0.150
and 0.003 to 0.045, respectively. The observed per-cell pmoA transcript levels for these
cultures are similar to those in M. trichosporium OB3b cultures (0.25 to 120.74) grown
in the same type of reactor (37). They are also comparable to other reported per-cell
pmoA transcript amounts under controlled (21, 44–46) and in situ (35, 36, 47) condi-
tions and reported per-cell methane oxidation rates (17, 22, 48, 49). Higher per-cell
pmoA transcript levels matched well with cultures with shorter RTs for both M. album
BG8 and M. parvus OBBP. Increased per-cell pmoA transcript amounts are hypothesized
to correspond to more active cells with increased activities. For the three aerobic meth-
anotroph cultures in Fig. 2, a strong positive correlation (Pearson’sR
2
= 0.91) between
per-cell pmoA transcript levels and per-cell methane oxidation rates was observed
across several orders of magnitude. Strong correlations were maintained when pure
methanotroph cultures were examined individually (Fig. S3). Cell amounts alone and
methanotrophic activity have been shown to be poorly correlated previously in metha-
notrophs (37, 50). Identical populations in terms of cell abundances can display vastly
different activities, which could explain the stronger relationship when looking at cell
amounts in conjunction with transcript amounts. The strong correlation observed for
per-cell pmoA transcript levels and per-cell methane oxidation rate is due to the regu-
lation of pMMO in response to available methane for oxidation. These results, spanning
three aerobic methanotroph species, including both type I and type II methanotrophs,
demonstrate that per-cell pmoA transcript levels may serve as a universal quantitative
biomarker of extant bacterial methanotrophic activity. To date, this correlation has
only been demonstrated under controlled lab conditions using pure cultures and
requires more robust testing in complex microbial bioreactors and ecosystems.
TABLE 1 Detailed operating conditions for M. album BG8 reactors
Retention time (days) Condition Time (days) Operational change Times when samples were collected
2.8 60.1 Steady state 0–15.2 Days: 12, 13,
a
and 15
CH
4
limitation 15.2–16.2 CH
4
off Hours after CH
4
off: 0.5, 1, 2,
a
8, 12, and 24
Recovery 16.2–17.2 CH
4
on Hours after CH
4
on: 8 and 24
5.7 60.1 Steady state 17.2–38.8 RT increase Day: 30
4.3 60.1 Steady state 0–15.2 Days: 12, 13,
a
and 15
O
2
limitation 15.2–16.2 O
2
off Hours after O
2
off: 0.5, 1, 2,
a
8, 12, and 24
Recovery 16.2–17.2 O
2
on Hours after O
2
on: 8 and 24
9.1 60.2 Steady state 17.2–38.8 RT increase Day: 30
a
Samples were selected for RNA-seq.
TABLE 2 Detailed operating conditions for M. parvus OBBP reactors
Retention time
(days) Condition Time (days)
Operational
changes
Days when samples
were collected
2.8 60.1 Steady state 0–19.5 18, 19
5.8 60.2 Steady state 19.5–36.2 RT increase 32, 34
4.3 60.2 Steady state 0–19.5 17, 18, 19
9.1 60.4 Steady state 19.5–44.0 RT increase 36, 43
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M. album BG8 reactor response to substrate limitation. At 15.2 days of operation,
effects of a 24-h methane and oxygen limitation on M. album BG8 cultures were explored.
The limitation period was followed by a 24-h recovery period in which membrane pres-
sures were turned back on (Fig. 3 and Fig. S2A and B). Methane limitation caused reactor
oxygen levels to increase from 5.5 to 9.0 mg L
21
due to the lack of incoming methane,
while biomass levels decreased by about half (Fig. 3A). The decrease in biomass was
reflected in both cell genome and pmoA mRNA amounts (Fig. 3B). Within 2 h of methane
limitation, genome copies were about a third of steady-state amounts, while pmoA mRNA
amounts decreased by about 3 orders of magnitude. During steady state, per-cell pmoA
transcript levels ranged from 9.02 to 11.60 transcripts per cell. A sharp decrease in the
pmoA transcriptional activity 0.5 h after the onset of methane limitation was observed,
reaching its minimum 8 h postlimitation, with a log
2
fold decrease of 8.2 in per-cell pmoA
transcript levels compared to average steady-state levels (Fig. 3C and Table S3). Per-cell
pmoA transcript levels stayed significantly depleted through the 24 h following the onset
of methane limitation. Resumption of methane saw sharp increases in pmoA transcript
amounts, with per-cell pmoA transcript levels approaching steady-state levels 24 h after re-
covery, while biomass (as both genome copies and dry cell weight) lagged (Fig. 3B and C
and Table S3).
In oxygen-limited reactors, a sharp decrease in biomass levels was also observed, which
continued into the recovery period (Fig. 3D). Steady-state pmoA transcript levels for the
4.3-day reactors ranged between 24.19 and 47.52 for the days preceding oxygen limitation.
During oxygen limitation, oxygen levels decreased from 4 to 2 mg L
21
and methane levels
increased from 1 to 3 mg L
21
. Genome copies dropped slowly throughout both the limita-
tion and recovery periods, while pmoA mRNA amounts decreased by 2 orders of magni-
tude 0.5 h after oxygen limitation, with a more gradual decline observed in the subsequent
hours during oxygen limitation (Fig. 3E). The decrease pattern in pmoA transcript amounts
was reflected in per-cell pmoA transcript levels, with a log
2
fold decrease of 4.3 in com-
pared to average steady-state levels (Fig. 3F and Table S3). The oxygen recovery period
also saw increases in pmoA transcript amounts which led to per-cell pmoA transcript levels
comparable to steady-state levels after 24 h following oxygen repletion (Fig. 3B and C and
Table S3).
The M. album BG8 biomarker response observed due to methane and oxygen limitation
differed compared to that in steady state. Genome copies in the methane-limited reactors
exhibited a sharp decrease within the first hour, with no further change observed through
24 h until recovery, when a small increase in genome copies was observed, while oxygen-
FIG 2 Steady-state per-cell pmoA transcript amounts and methane oxidation rates for aerobic
methanotrophs. Data are averages from individual reactors from distinct sampling dates. Error bars
represent standard deviations of biomarker amounts (yaxis) from replicate reactors. Power law trend
and R
2
value are shown. M. trichosporium OB3b data were obtained from the work of Tentori and
Richardson (37).
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limited reactors exhibited a steady decrease throughout both the limitation and recovery
periods (Fig. 3B and E). Both methane and oxygen limitation had the largest decrease of
pmoA transcript levels in the first hour, with a larger overall decrease observed with meth-
ane limited. Differences between per-cell pmoA transcript levels during both methane and
oxygen limitation and their preceding steady-state levels were statistically significant
(P,0.001) (Table S4). Both sets of reactors reached per-cell transcript levels comparable to
steady-state averages within the 24-h recovery period. Differences in per-cell pmoA tran-
script levels were statistically significant during methane limitation and recovery (P,0.01)
and during oxygen recovery and the preceding limitation period (P,0.05) (Table S4). The
impact of oxygen limitation may have been less than that of methane limitation, as oxygen
was present in the reactor influent. Similar pmoA expression patterns have been observed
in mixed-consortium bioreactors, where cultures grown under feast conditions showed 2-
to 3-fold per-cell pmoA transcript decreases within 1 h of famine conditions (51). Rodríguez
et al. (51) also observed that per-cell pmoA transcripts of methanotroph cultures regularly
exposed to recurring feast-famine conditions were not affected by the onset of famine,
seeing no change in the first 12 h. Overall pmoA mRNA per-cell levels observed in the cur-
rent study were orders of magnitude higher than in the study by Rodríguez et al. (0.00005
to 0.002) (51) and in line with those of M. trichosporium OB3b cultures (37, 52) and
Methyloprofundus sedimenti (21). The observed decrease in per-cell pmoA transcripts during
FIG 3 M. album BG8 reactors during substrate limitation and recovery. (A) 2.8-day RT reactor response during CH
4
limitation; (B) 2.8-
day RT reactor genome and pmoA mRNA amounts during CH
4
limitation; (C) 2.8-day RT reactor pmoA mRNA copies per cell during
CH
4
limitation; (D) 4.3-day RT reactor response during O
2
limitation; (E) 4.3-day RT reactor genome and pmoA mRNA amounts during
O
2
limitation; (F) 4.3-day RT reactor pmoA mRNA copies per cell during O
2
limitation. Data are means 6standard deviations from
triplicate reactors. Asterisks indicate samples selected for RNA-seq. Data following RT switch are not shown.
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the first hours of methane limitation suggest a fast pmoA regulation in response to meth-
ane levels. This is also supported by the increase observed in per-cell pmoA transcripts
when methane was available during the recovery period, where a response occurred in
the first 8 h and reached steady-state levels after 24 h (Fig. 3C). The observed rapid
response of methanotrophs to changing methane and oxygen conditions suggests that
they can quickly adapt to changes in local environmental conditions.
In addition to cell amounts decreasing, the observed changes in biomass could be
due to decreases in protein mass, as starvation under aerobic conditions has been
demonstrated to induce protein biomass loss in methanotrophs (17). Rapid changes at
the transcript level due to changes in local conditions are well documented for aerobic
methanotrophs. The “copper switch,”a well-established link between copper levels
and expression of pMMO or sMMO, has been shown to occur in the order of minutes
to hours (23, 53, 54). Aerobic methanotrophs have also been shown to regulate expres-
sion of methane oxidation pathways in response to the type and amount of carbon
source available (e.g., methane versus other one-carbon compounds) (55–57). This sug-
gests that decreased methane amounts during periods of limitation would lead to a
decrease in pmoA expression and a decrease in pmoA transcripts per cell and the oppo-
site effect during the recovery period. Rapid (,2-h) responses from methanotrophs to
starvation and recovery periods when grown in different biofilters and stirred tank
reactors have been observed (51, 58). Rodríguez et al. grew methanotroph reactors
under different operational methods, feast-famine growth, and continuous growth and
examined the influence of operational period on performance (methane consumption)
and pmoA expression levels. Following a famine period, reactors under both opera-
tional methods were able to recover to their prelimitation performance within 2 h;
however, the pmoA expression levels were influenced by operational mode, with a
more drastic decrease observed in pmoA expression levels for the continuous-growth
reactors (51). However, different trends have been observed in the methanotroph M.
sedimenti, for which short-term methane starvation periods led to increased pmoA
expression throughout the starvation period, with the opposite trend observed during
recovery (21). Differing expression patterns between methane oxidation and the meth-
anol dehydrogenase (MDH) genes mxaF and xoxF have been observed during starva-
tion, suggesting independent regulation (21). The differences in expression observed
between methanotrophs could serve as different survival strategies during starvation
periods.
M. album BG8 transcriptomic samples and gene expression response to short-
term substrate limitation. Transcriptome sequencing (RNA-seq) samples from M. album
BG8 under short-term substrate limitation yielded 170.4 million reads across the 8 multi-
plexed samples from the four conditions (Table S5). Gene counts showed agreement after
normalization, with medians consistent across samples and between biological replicates
(Fig. S4). Biological replicates were examined using principal-component analysis (PCA) of
normalized logarithmic transformed read counts using DESeq2 (59). Similarity was
observed between duplicate biological replicates for the substrate-limiting conditions.
Steady-state samples showed less uniformity due to one replicate from the 4.2-day RT (Fig.
S5). Despite the variability observed in PCA of the M. album BG8 transcriptome samples
under steady state, samples were not considered outliers, as no significant differences in
transcripts were detected. Coverage and normalized counts were similar across replicates,
and replicates for all conditions were included in subsequent analyses.
Collectively across all transcriptomes, expression was observed for 3,931 out of 3,984
(98.7%) of predicted protein-coding genes in the published M. album BG8 genome (60).
Significant differential gene expression (DGE) (log
2
fold change [FC] .j1.0 j; adjusted
Pvalue [P
adj
],0.05) was observed for CH
4
-andO
2
-limited samples compared to corre-
sponding steady-state samples (Table S6 and Data Set S2). Methane limitation and oxygen
limitation resulted in 444 and 282 genes, respectively, with significant DGE compared
to reference steady-state samples. Reference steady-state samples had similar expres-
sion profiles regardless of 2.8- or 4.2-day RT, with only 4 genes showing significant
DGE. EggNOG 5.0 was used to categorize genes showing DGE between substrate-limited
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and corresponding steady-state samples, categorizing 74% and 81% of differentially
expressed genes of the methane- and oxygen-limited samples, respectively (Fig. 4).
Both CH
4
-andO
2
-limited conditions triggered upregulation of several of genes related
to cell motility, flagella, and chemotaxis (identifier in Clusters of Orthologous Groups [COG]
database: N) (Fig. 4 and Data Set S2). Upregulation of motility genes has been linked to nu-
trient limitation, with motile cells seeking more favorable conditions (61). In methano-
trophs, growth conditions influence expression of motility and adhesion genes. Similar
expression patterns have been observed in M. album BG8 and Methylocystis sp. strain
Rockwell grown with methanol (62) in which flagellum genes were upregulated. In this
study, both limitation of CH
4
and limitation of O
2
resulted in a strong consistent downregu-
lation of translation, ribosomal structure, and biogenesis (COG: J) genes, with effects on ri-
bosomal protein genes more pronounced in methane limitation, while oxygen limitation
mostly affected translational genes (Fig. 4 and Data Set S2).
The changes in expression observed, with an increase in cell motility, posttransla-
tional modifications, and signal transduction genes and a decrease in carbohydrate,
amino acid, and lipid metabolism and transport, and translation genes, are similar to
those observed for M. album BG8 grown with methanol compared to methane (62).
For all conditions, genes involved in methane oxidation and methanol oxidation were
among the most highly expressed genes (16.6 to 28.9% of all transcripts [Data Set S2]), fol-
lowing expression trends observed in aerobic methanotrophs (63–65). The general trend
was downregulation in C
1
metabolism genes upon short-term limitation. Exceptions were
upregulation of alcohol dehydrogenase gene xoxF under both conditions and formate de-
hydrogenase (fdh)specifically during methane limitation. However, the C
1
metabolism
gene transcript remained among the most highly abundant reads detected even during
stress. A period of substrate limitation longer than 2 h might be required to observe an
effect due to elevated starting transcript amounts for these pathways and the half-life of
RNA. Partially degraded RNAs may still being “readable”by the RNA-seq method, unlike
with qPCR, in which full-length transcripts are required for detection.
A strong effect was observed in genes involved in energy production and conver-
sion (COG: C) (Fig. 4). For methanotrophs, these changes in expression of genes for
energy production and conversion are hypothesized to be in response to the available
substrate, to compensate for differences in energy available and minimize effect on
core metabolic pathways (66). A visual representation of the response of central meta-
bolic pathway genes and other genes of interest to methane and oxygen limitation is
FIG 4 Classification of significant differential gene expression (DGE) in M. album BG8, based on COG
classification from the eggNOG 5.0 database. Positive and negative values on the xaxis indicate
numbers of genes in that COG category that were upregulated and downregulated, respectively,
compared to steady-state conditions. Unclassified, category S (“Function Unknown”) and categories
with fewer than two genes with DGE are not shown.
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shown in Fig. 5. NADH oxidoreductase and cytochrome genes were upregulated under
methane-limited conditions, while ATP synthases were downregulated, with stronger
effects observed under CH
4
limitation. Upregulation of cytochrome oxidase genes in
response to O
2
limitation was observed, and such upregulation has been previously
observed in Methylomicrobium buryatense 5GB1C (20); however, a stronger response
FIG 5 Changes in central metabolic pathways in M. album BG8 during short-term substrate limitation of CH
4
(top) and O
2
(bottom).
Upregulated (green) and downregulated (purple) pathways and pathways with no change (gray) compared to steady-state conditions
are highlighted. Multiple colors indicate that different genes in that pathway were both up- and downregulated. Abbreviations and
corresponding intermediates and compounds are provided in Table S7.
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was observed for CH
4
limitation. Different carbon sources have been found to affect
transcriptional responses of both alpha- and gammaproteobacterial methanotrophs
(55, 67).
M. album BG8 upregulated pentose phosphate (PP) pathway genes and electron trans-
port chain (ETC) genes, namely, NADH oxidoreductase (nuo) and cytochrome genes, to
adapt methane limitation while downregulating other metabolic activity (Fig. 5 and 6).
During oxygen limitation, Entner-Doudoroff (ED) pathway genes, in addition to PP pathway
genes, were upregulated, while Embden-Meyerhof-Parnas (EMP; glycolysis) pathway genes
were downregulated and electron transport chain genes saw both slight up- and downreg-
ulation. Genes involved in oxidation of methane (pmo)andmethanol(mxa) were generally
downregulated under both conditions. A homolog of methanol dehydrogenase, XoxF, was
highly upregulated under both limiting conditions. XoxF-type methanol dehydrogenases,
however, are lanthanide dependent (68), and reactor medium was unchanged, indicating
that this could be a survival response upon nutrient limitation or to shift metabolism entirely
during stress conditions. These could be potential strategies for maintaining activity and
growth upon substrate limitation. Research on M. buryatense 5GB1 and M. album BG8
grown on methanol suggests that electrons obtained from methanol dehydrogenase are
directly transferred to cytochromes and the electron transport chain, bypassing the electron
requirements from NADH oxidoreductase complex for ATP production (67). Some studies
have shown that type I methanotrophs potentially perform fermentation-based methano-
trophy, in which methane-derived formaldehyde can be used for formation of formate, ace-
tate, succinate, lactate, and hydroxybutyrate under low-oxygen conditions (69). In
Methylomicrobium alcaliphilum 20Z, low oxygen induced increased expression of EMP
genes, predicted fermentation pathway enzyme genes and O
2
carrier hemerythrin, and
decreased expression of NADH:ubiquinone oxidoreductase and cytochrome coxidase in
addition to detection of possible fermentation products and H
2
(69). The genome of M. bury-
atense 5GB1 also contains homologs of enzymes required for fermentation, and its metabo-
lism under O
2
limitation is a combination of fermentation and respiration (20). Another
potential strategy during substrate limitation is use of internal carbon storage. M. album
BG8 has genes for glycogen biosynthesis, just as does “Candidatus Methylacidiphilum fumar-
iolicum”SolV from phylum Verrucomicrobia, which consumes stored glycogen during peri-
ods of substrate limitation (19). In this study, periods of both methane and oxygen limitation
led to significant downregulation of glycosyltransferase and glycogen synthase genes and
an upregulation in glycogen debranching and cleaving genes (COG: G), which are poten-
tially involved in glycogen synthesis and degradation, respectively (Fig. 6).
Short-term substrate limitations led to transcriptional changes in ETC enzyme
genes, preference for the PP pathway, and, in the case of O
2
limitation, upregulation of
ED pathway genes. These responses show potential strategies where pathways are
shifted to use internal carbon reserves and, potentially, fermentation for survival. In
addition to the changes in carbon metabolism, observed changes in gene expression
in both methane- and oxygen-limited samples were indicative of methanotrophs with
decreased activity (70), and short-term substrate limitation was sufficient to elicit a
stress response. DGE genes and additional genes of interest grouped by COG for sub-
strate-limited samples are shown in Fig. 6.
Additionally, both limitation conditions had downregulation of the carbon storage reg-
ulator, csrA,andsignificant upregulation of sigma factor rpoE, an expression pattern indica-
tive of stress response (71). DGE was observed for other genes indicative of stress response
as seen for stressed M. album BG8 grown with methanol (67) (Fig. 6). Methane limitation
led to upregulation of oxidative stress genes, including superoxide dismutase (log
2
FC = 1.8; P
adj
,0.05), peroxiredoxin (log
2
FC = 2.5; P
adj
,0.05), and thioredoxin (log
2
FC = 1.6; P
adj
,0.05) genes, likely to deal with increased oxidative stress as dissolved-oxy-
gen levels increased after methane limitation (Fig. 6). Under O
2
-limited conditions, a 2-fold
increase was observed for hemerythrin, a putative O
2
-scavengingprotein(72)whichisup-
regulated in Methylomicrobium buryatense 5GB1C (20) and Methylomicrobium alcaliphilum
20Z (69) under conditions of oxygen limitation. Additional stress indicators were
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FIG 6 Gene expression profiles of M. album BG8 under short-term substrate (O
2
and CH
4
) limitation compared to steady-state
conditions. Statistically significantly differentially expressed genes (log
2
FC .j1.0 j,P
adj
,0.05) are indicated by asterisks. Genes
are grouped by orthology and/or function. Subset of genes shown; see Data Set S2 for complete differential expression analyses.
Biomarker Response of Methanotrophs to Limitation Microbiology Spectrum
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downregulation of DNA polymerase genes and upregulation of competence and DNA-pro-
tecting protein genes (COG: L) (Fig. 6). In addition to transcriptional regulation, shifts in
core metabolic processes could be regulated via translational and posttranslational mecha-
nisms as well (66).
The 2-h substrate limitation period was enough to observe a transcriptional response in
M. album BG8 under both conditions. Experimental and modeling results have shown that
transcriptional responses in bacteria due to substrate limitation are fast, being elicited
within 1 h, while translational changes may continue for more than 10 h (73). When com-
pared to steady-state levels, RNA-seq data showed pmoA transcript amounts decreased
about 2-fold during substrate-limiting conditions. However, the effects were not as drastic
as observed in reverse transcription-qPCR (;100-fold decrease). The observed difference
could be due to partially decayed transcripts being read in RNA-seq, whereas for reverse
transcription-qPCR, only the nondegraded full pmoA target region would be amplified and
detected.
This work highlights mRNA biomarkers as indicators of methanotrophic activity as
well the transcriptomic response of M. album BG8 to short-term substrate limitations,
demonstrating a fast response to dynamic conditions and potential strategies for
growth under periods of limited substrate availability. The results presented suggest a
cross-genus, potentially universal trend between steady-state per-cell activity and per-
cell pmoA transcript levels in aerobic bacterial methanotrophs across several orders of
magnitude. Universal trends would only be possible if these correlations are explored
in anaerobic, bacterial methanotrophs from phylum NC10 and Verrucomicrobia. This
work holds promise for assessing methanotroph growth and activity in diverse envi-
ronments and informs operational strategies in biotechnological applications of meth-
anotrophs, for example, in biorefinery scenarios where biogas is upcycled into more
valuable bioproducts.
MATERIALS AND METHODS
Bacterial strains, reactor design, and experimental conditions. Experiments were conducted at
25°C using pure cultures of M. parvus strain OBBP (NCIMB 11129) and M. album strain BG8 (NCIMB
11123) grown on nitrate mineral salts (NMS) medium (74). Prior to inoculation of reactors, methanotroph
cultures were grown in crimp-capped serum bottles as previously described (37). Bioreactors consisted
of modified 1-L Pyrex medium bottles (Corning, Corning, NY), with a 0.80-L liquid volume and 0.27-L
headspace volume and with two sets of silicone tubing serving as membranes for bubbleless delivery of
CH
4
($99.5% purity; Airgas, Radnor, PA) and O
2
($99.99% purity; Airgas), as previously described (37).
Reactors were operated as chemostats, continuously provided NMS medium, and RTs were determined
by measuring reactor effluent, as previously described (37). During periods of substrate limitation for M.
album BG8 reactors, membrane pressures were disconnected from gas tanks and opened to room
atmosphere. Two sets of triplicate reactors of M. album BG8 (Table 1) and M. parvus OBBP (Table 2) cul-
tures each were started concurrently with initial RTs of 2.8 and 4.3 days.
Reactors were operated under the initial conditions until methane and biomass levels stabilized,
considered steady-state operation. This initial periods were approximately 15 days (3 to 5 retention
times) for M. album BG8 reactors and 19.5 days (.4 RTs) for M. parvus OBBP reactors. Following the
steady-state period, the methane and oxygen supplies were shut off in the M. album BG8 2.8-day and
4.3-day reactors, respectively, for a 24-h period of substrate limitation. After 24 h, methane and oxygen
were turned back on for a 24-h recovery period, before the M. album BG8 2.8- and 4.3-day RT reactors
were transitioned to longer retention times of 5.8 and 9.1 days, respectively. M. album BG8 reactors
were operated to reach a new steady state (2.5 to 3 RTs). After 19.5 days (.3 RTs), M. parvus OBBP reac-
tors were transitioned to retention times of 5.8 and 9.1 days and operated until steady-state operation
was achieved. Diagrams showing reactor operation and sampling times of both cultures are provided in
Fig. S2.
Methane, oxygen, biomass sampling, and methane oxidation rate measurements. Reactor meth-
ane, oxygen, and microbial biomass concentrations were monitored throughout operation. Methane
and oxygen concentrations were determined from reactor headspace measurements using gas chroma-
tography with a thermal conductivity detector (GC-TCD) and Henry’s law coefficient, and microbial bio-
mass was monitored via readings for optical density at 600 nm (OD
600
) and converted to cell biomass
(dry weight) as previously described (37). Biomass samples for nucleic acid extraction were collected on
selected dates during both steady-state and substrate limitation periods (Tables 1 and 2). Methane oxi-
dation rates were determined using reactor methane and biomass data, available organism kinetic pa-
rameters (12, 40, 75), and a mechanistic model describing the membrane bioreactor system (37). Reactor
methane oxidation rates (milligrams of CH
4
per liter per day) were converted to obtain methane oxida-
tion rates per unit volume, and cell normalized methane oxidation rates were obtained using
Biomarker Response of Methanotrophs to Limitation Microbiology Spectrum
May/June 2022 Volume 10 Issue 3 10.1128/spectrum.00003-22 12

determined pmoA gene copies and pmoA copies per genome for each organism (1 copy for M. album
BG8 [60] and 2 copies for M. parvus OBBP [76]).
Nucleic acid extraction, cDNA synthesis, and qPCR analyses. Sample collection, extraction, storage,
and processing were done as previously described (37). Nucleic acid quality assurance/quality control (QA/
QC) was performed at the Cornell Biotechnology Resource Center (BRC). Populations (genome copies) and
activities (pmoA transcripts) of M. parvus OBBP and M. album BG8 cultures were monitored via qPCR and
reverse transcription-qPCR, respectively, targeting gene pmoA (subunit A of pMMO) using previously pub-
lished general aerobic methanotroph primer set A189F/Mb661R (24, 77). Luciferase (luc)DNAandmRNA
(Promega, Madison, WI) spike-ins were used as internal standards to correct for losses during extraction and
reverse transcription (78), using previously described methods (37). All qPCRs were performed in triplicate
20-
m
L reaction mixtures using Luna universal qPCR master mix (New England Biolabs) on an iCycler IQ multi-
color real-time detection system (Bio-Rad), with contamination checks and verification of correct qPCR prod-
uct performed as previously described (24, 37, 78). Methanotroph cell amounts (as genome copies) were
determined via qPCR from pmoA gene amounts and pmoA copies per genome for each organism as
described above. Biomarker pmoA transcript levels were determined via reverse transcription-qPCR on cDNA
and normalized by genome copies to obtain per-cell pmoA transcript amounts. Differences between pmoA
transcript amounts per cell for steady state, substrate limitation, and recovery conditions were evaluated with
the Mann-Whitney U test using R studio software (v3.4.2).
RNA sequencing, assembly, and differential gene expression. RNA-seq was performed on dupli-
cate M. album BG8 samples at steady state (day 13) and 2 h after substrate limitation (Table 1 and Fig.
S2). Samples were submitted to Cornell University’s Transcriptional Regulation & Expression Facility
(TREx) for quality checks, library preparation, and RNA sequencing. RNA integrity was determined using
a fragment analyzer (Advanced Analytical, Santa Clara, CA). Using a total RNA input of 500 to 1,000 ng,
rRNA was subtracted by hybridization from total RNA samples using the NEBNext rRNA depletion kit for
bacteria (New England Biolabs). TruSeq-barcoded RNA-seq libraries were generated with the NEBNext
Ultra II directional RNA library prep kit (New England Biolabs) following the manufacturer’s instructions.
Libraries were quantified with a Qubit 2.0 (double-stranded DNA [dsDNA] high sensitivity (HS) kit;
Thermo Fisher), and size distribution was determined using a fragment analyzer (Advanced Analytical)
prior to pooling. Libraries were sequenced on an Illumina HiSeq X Ten system (Illumina, Inc., San Diego,
CA) with 2 150-nucleotide (nt) paired-end reads, generating at least 4 million reads per library.
Illumina pipeline software was used for base calling, Sequenced reads were trimmed for 39adaptor
sequence and low-quality sequence and filtered to remove reads of ,50 nt with TrimGalore v0.6.0 (79),
a wrapper for Cutadapt (80), and FastQC (81). Processed reads were mapped to the M. album BG8 ge-
nome using STAR v2.7.0e (82) using–quantMode GeneCounts to generate raw counts per gene. Raw
counts were analyzed in R using SARTools (83) and DESeq2 v1.26.0 (59) to generate normalized counts
and for statistical analysis of differential gene expression (DGE). Significance in differential expression
was considered at a log
2
fold change (FC) of .j1.0 jand false-discovery rate (FDR)-adjusted Pvalue
of ,0.05.
Genes with significant DGE during substrate limitation compared to steady-state conditions were
mapped to functional identifiers in the Clusters of Orthologous Groups (COG) database using eggNOG
5.0 (84). Remaining unclassified genes, or genes classified as “Function Unknown,”were further assessed
via the National Center for Biotechnology Information Basic Local Alignment Search Tool (NCBI-BLAST;
https://blast.ncbi.nlm.nih.gov/Blast.cgi). M. album BG8 RNA-seq data were submitted to the NCBI Gene
Expression Omnibus (GEO) database under accession number GSE188821.
SUPPLEMENTAL MATERIAL
Supplemental material is available online only.
SUPPLEMENTAL FILE 1, PDF file, 0.5 MB.
SUPPLEMENTAL FILE 2, XLSX file, 0.4 MB.
ACKNOWLEDGMENTS
We thank Jeremy Semrau for providing the methanotroph cultures and Jen K. Grenier,
Ann Tate, and Faraz Ahmed (Cornell TREx) for their help with RNA-seq. We also acknowledge
helpful comments and input from colleagues Nan Wang and Jingyi Wu.
The manuscript was written through contributions of all authors. All authors have
given approval for the final version of the manuscript.
This work was supported by the David R. Atkinson Center for a Sustainable Future
(ACSF) Academic Venture Fund (AVF) program and a Cornell Sloan Graduate Fellowship
to E.F.T.
We declare no competing financial interests.
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