Biological valorization of methane and nitrogen gas-derived ammonia via methanotrophic bacteria for gut-beneficial nutrients

Domain: biology
Published: 2026-03-11
Editorial outcome: minor_revision

Abstract
The in-situ upcycling of decentralized methane and nitrogen gas (N )-derived ammonia via
2
methanotrophic bacteria is highly attractive. However, the toxic intermediate generated from
ammonia oxidation significantly inhibits cell growth, thereby hindering efficient bioproduction.
Herein, by integrating transcriptomic analysis, we develop rational metabolic engineering
strategies and an optimized fed-batch fermentation to enhance ammonia utilization in a
methanotrophic bacterium of Methylotuvimicrobium sanxanigenens. The modified M.
sanxanigenens overexpressing hydroxylamine reductase efficienStly co-assimilates methane and
ammonia for cell protein synthesis, with an 18-fold increase in productivity. The resulting
methanotrophic cell protein (MCP) not only exhibits an Rideal essential amino acid profile but also
contains bioactive nutrients, including polysaccharides and peptides. Oral administration of this
nutritional MCP significantly ameliorates colitis symptoms in male mice by attenuating
inflammatory progression and restoring the intestinal barrier. Moreover, MCP treatment maintains
gut microbiota homeostasis and promotes the excretion of beneficial metabolites, thereby
protecting the intestinal microeInvironment. Hence, this study provides a promising biological
approach for the local bioR-valorization of decentralized CH
4
and air into functional feed additives.
This biotechnology not only facilitates advancements in developing carbon-negative gas-to-value
pathways but also drives green transformations in animal husbandry by reducing the use of
antibiotics and vaccines.

Introduction
China has abundant decentralized methane (CH ) derived from fossil fuel extraction and organic
4
waste treatment processes.1,2 However, it is difficult and costly to collect these CH resources via
4
pipelines and upgrade them for natural gas production. Thus, decentralized CH is often combusted
4
for low-value heat and power to meet the energy demands of surrounding factories, ultimately
resulting in the complete waste of carbon resources. Aerobic methanotrophic bacteria, however,
can utilize CH to synthesize single cell protein as an alternative feed additive.3 This approach may
4
not only valorize decentralized CH but also alleviate protein shorStages—a critical issue given that
4
S
more than 80% of China’s soybean and fishmeal supplies rely on imports, totaling over 100 million
E
tons annually.4 This context underscores the urgent need Rto develop efficient biological approaches
P
for valorizing decentralized CH₄ into protein feed, thus addressing both carbon resource waste and
N
protein supply challenges.
I
E
L
This biological upcycling of decentralized CH is technically feasible, as mature technologies exist
4
C
for supplying key nutrients. As oIne of the most widely used nitrogen sources in biomanufacturing,
T
ammonia can be produceRd from nitrogen gas (N
2
) via an efficient and environmentally friendly
A
photo-electrocatalytic (PEC) process.5 Cryogenic distillation, a well-established technology in the
chemical industry, separates high-purity N from air. Under the assumed conditions
2
(Supplementary Note 1), this process also co-produces high-purity oxygen gas (O ).6 Therefore,
2
simultaneous utilization of O generated from both air separation unit (ASU) operations and the
2
PEC process enables maximal O utilization, as O serves as a crucial substrate for methanotrophic
2 2
bacteria.3 Detailed balanced reactions and corresponding calculations are provided in
Supplementary Note 2 and Supplementary Note 3. Moreover, the single cell protein produced can
be processed into microbial nutrients and formulated into animal feed, directly supporting nearby
livestock farms. This integrated supply chain—where air-derived O₂ and green ammonia support
CH₄-based microbial protein synthesis—lays a solid technical foundation for the sustainable, local
upcycling of decentralized CH₄.

However, several vital issues must be addressed to fully realize this prospect. First, methanotrophic
bacteria are not natural consumers of ammonia. Notably, it is widely reported that ammonia is
partially oxidized by methane monooxygenase (MMO) to form a highly toxic intermediate,
hydroxylamine—a compound that significantly impairs the growth of aerobic methanotrophic
bacteria.7 Given the difficulty in modifying MMO’s selectivity toward ammonia without
compromising its methane oxidation activity, there is an urgent need to investigate the mechanisms
underlying deficient ammonia utilization by integrating omics analysis. This serves as a
S
prerequisite for designing rational strategies to overcome metaSbolic bottlenecks in hydroxylamine
E
detoxification. Furthermore, although several studies have shown that methanotrophic bacterium-
R
derived cell protein promotes intestinal health iPn fish,8,9 its biofunctions and associated
mechanisms remain underexplored in standarNdized animal models. This currently restricts its
I
application as a bulk protein substitute—im peding its ability to compete with traditional feeds and
E
gain acceptance from markets and coLnsumers.10 These challenges greatly hamper endeavors to
C
establish proof of concept for the in-situ valorization of decentralized CH₄.
I
T
R
In this study, we seleAct Methylotuvimicrobium sanxanigenens NG09 (CGMCC No. 30951)—a
methanotrophic bacterium isolated from rice paddies with a high growth rate—as a CH -utilizing
4
cell factory.11 To clarify the mechanism of its growth inhibition by ammonia, we perform a
transcriptomic analysis to identify key enzymes involved in hydroxylamine detoxification. Based
on these findings, we rationally construct the engineered strain SO01 overexpressing
hydroxylamine reductase to enhance its growth with ammonia as the sole nitrogen source (Figure
1). We further develop a fed-batch fermentation strategy to match the ammonia production
capacity of upstream N fixation with the downstream growth demands of strain SO01. To expand
2
the applications beyond a mere protein substitute, we investigate the effects of the produced
microbial nutrients on attenuating dextran sulfate sodium (DSS)-induced colitis in mice.
We observe that these microbial nutrients also protect the intestinal microenvironment in mice, as
evidenced by the recovery of gut microbiota diversity and the promotion of prebiotic excretion.
Our work thus highlights the prospect of biovalorizing decentralized CH and air into value-added
4
functional animal nutrients.

Results
Uncovering ammonia-induced toxicity mechanism helps to rationally modify
methanotrophic chassis
Methanotrophic cultivation typically relies on nitrate as nitrogen Ssource,12,13 primarily due to the
S
severe growth inhibition associated with ammonia utilization. Given that the cost of nitrate salts is
E
approximately twice that of ammonia,14 it is thus crucial Rto endow methanotrophic bacterial chassis
P
with enhanced ammonia utilization capacity. Although ammonia is the preferred nitrogen source
N
for protein biosynthesis in most microorganisms, it is partially oxidized by MMO to form toxic
I
hydroxylamine, which significantly hampers cell growth. We firstly tested the growth ability of M.
E
sanxanigenens NG09 with ammonia (for the remainder of this manuscript, molecular ammonia
L
(NH ) and ionic ammonium (NHI+) salt are collectively referred to as “ammonia” for consistency).
3 4
C
Similar to other methanRotrophic species,15 M. sanxanigenens NG09 cultured with ammonia
T
exhibited a 12-h longer lag phase than when grown with nitrate (Figure 2a)—a nitrogen source
A
widely used in methanotrophic bacteria cultures. This indicated that the cells may experience an
adaption period for utilizing ammonia. Additionally, M. sanxanigenens NG09 firstly utilized
ammonia when both two nitrogen sources existed, and nitrate uptake was observed only after
ammonia depletion (Supplementary Figure 1 and Supplementary Note 4).

To clarify the underlying mechanisms of ammonia preference and related growth retardation, we
sought to find evidence from whole-genome transcriptional responses of M. sanxanigenens NG09
grown in the exponential phase. This approach enables the identification of key detoxification-
related enzymes, which in turn facilitates the development of a rational genetic modification
strategy to enhance cellular ammonia utilization. In the PCA analysis, the principal component 1
explained 95% of the variation (Figure 2b). High correlations among samples within groups,
together with weak correlations among different groups (Supplementary Figure 2a–b), indicated
that the type of nitrogen source was the prominent cause of changes in gene transcription.
Compared with growth with nitrate, 121 and 150 genes were significantly up- and down-regulated
in the ammonia culture (Supplementary Figure 2c–d), respectively.

As ammonia toxicity is proposed to be highly relevant to the formation of hydroxylamine, we then
focused on the 45 differentially expressed genes involved in the nitrogen metabolism. The genes
encoding nitrate reductase (nar), nitrate transport proteins (nrtASBC), and nitrite reductases (nirBD
and nir) were all repressed in cultures with ammonia (Figure 2c). This explained the phenomenon
of
blocked nitrate uptake when ammonia existed. APdditionally, N fixation, urea and cyanate
2
assimilation pathways were all inactivated (SuppNlementary Data 1). These changes were consistent
I
with the fact that ammonia is the preferred nitrogen source with the lowest energy and reducing
E
power demand.16 However, when M. sLanxanigenens NG09 was cultured with ammonia, the genes
C
encoding hydroxylamine reductase (har) and dehydrogenase subunits (haoAB) were significantly
I
T
up-regulated by 3.9-, 3.8- and 3.3-fold, respectively. This transcriptional response indicates that
R
the strain was subjecteAd to hydroxylamine-induced toxicity, a conclusion further supported by the
detection of 70.5 μM hydroxylamine per unit cell density (OD ) in the ammonia-fed culture
600
(Supplementary Figure 3). By contrast, no hydroxylamine was detected in the nitrate cultures,
which can be attributed to the absence of hydroxylamine-generating steps in the nitrate
assimilation pathway.17,18 Correspondingly, M. sanxanigenens NG09 accumulates 3.34 mg of
NO −-N L−1 in the ammonia-based cultures, an approximately 10-fold higher than that measured
2
in the nitrate-grown cultures (0.33 mg NO −-N L−1, Supplementary Figure 4). Together, the
2
transcriptomic analysis results confirmed pronounced impacts on the nitrogen metabolic network
of M. sanxanigenens NG09 when the nitrogen source was switched to ammonia.

We further designed metabolic engineering strategies to redirect hydroxylamine flux. The haoAB-
deficient strain showed severely impaired growth with ammonia (Supplementary Figure 5 and
Supplementary Note 5), indicating that hydroxylamine dehydrogenase is important for
detoxification. Additionally, as nitrite reductases were all repressed under ammonia culture, nitrite
accumulated due to enhanced hydroxylamine oxidation, thus hampering cellular metabolic
activities. However, negligible improvement of cell growth was observed by overexpressing
hydroxylamine dehydrogenase or two nitrite reductases (Supplementary Figure 6 and
Supplementary Note 6). Moreover, in order to alleviate the growth retardation, we also attempted
to regulate intracellular ammonia availability by eliminating the transporters. Nonetheless,
knockout of amtA and/or amtB did not notably affect the Slag phase and growth rate of the
engineered strains grown with ammonia. The NH +-N level exhibits no significant differences (p >
4 R
0.05) between the wild type and amt-knockout Pstrains, indicating that the growth of M.
sanxanigenens NG09 is not limited by amNmonia transport (Supplementary Figure 7 and
I
Supplementary Note 7). This result dem onstrates that knockout of the putative ammonia
E
transporters (AmtA and AmtB) doesL not affect the growth of M. sanxanigenens NG09 with
C
ammonia.

Another strategy is toA upregulate the transcriptional level of hydroxylamine reductase, which
catalyzes the direct hydroxylamine reduction to ammonia. On the basis of per mole hydroxylamine
recycled, the reducing power demand is calculated to be only one third of that in the NO −-to-NH
2 3
reduction. The araC-P regulatory system was established to control the overexpression of har.
BAD
The inducer arabinose exerts no significant impact on growth at concentrations below 0.5 g L−1
and is not assimilated by methanotrophic bacteria.19 Given its superior growth performance with
ammonia (Figure 2d−e), M. sanxanigenens SO01 was selected for subsequent cultivations, with
arabinose added at 0.1 g L−1 to induce har expression. When M. sanxanigenens SO01 was cultured
with ammonia and supplemented with 0.1 g L−1 arabinose to induce har expression, the
hydroxylamine concentration accumulated to 44.6 μM per unit OD . This level was 37.9% lower
600
than that in the ammonia-fed culture without L-arabinose supplementation (Figure 2f and
Supplementary Figure 8). The significant decreased hydroxylamine accumulation directly
supports that har overexpression mitigates hydroxylamine-induced toxicity in methanotroph cells.
In contrast, arabinose concentrations of 0.25 and 0.4 g L−1 impose an enhanced metabolic burden,
leading to cellular resource reallocation and slowed cell growth as a compensatory adaptation
(Figure 2d).20 Consequently, these higher concentrations fail to yield significant improvements in
har expression and even impair the growth of M. sanxanigenens SO01. In summary, the ammonia-
based growth capacity of M. sanxanigenens is enhanced by reinforcing direct hydroxylamine
reduction.

Developing ammonia fed-batch strategy to boost production of nutritional cell protein
In addition to metabolic engineering strategies, it is Pnecessary to regulate ammonia transport by
controlling its concentration, which has beenN reported to strongly influence the growth of
I
methanotrophic bacteria.7 We firstly tested a mmonia tolerance capacity of M. sanxanigenens SO01.
Pronounced growth inhibition of the hLar-overexpressing M. sanxanigenens SO01 occurred when
ammonia concentration reached 20 mM or higher (Supplementary Figure 9 and Supplementary
Note 8). However, more ammonia is required to achieve substantial cell protein production without
impairing growth. SuAbsequently, a fed-batch strategy was established, and the cell density
increased by nearly 70% when ammonia concentration did not exceed 10 mM (Supplementary
Figure 10), consistent with the results of ammonia tolerance test. In contrast, a rapid growth decline
occurred at 36 h in the high-dose cultivation. Therefore, further fed-batch cultivation was
conducted to control the ammonia concentration below 10 mM.

N -derived ammonia was used in M. sanxanigenens cell protein production. M. sanxanigenens
2
exhibited similar growth on N -derived ammonia in comparison to a commercial ammonia agent
2
(Supplementary Figure 11). Additionally, when M. sanxanigenens culture was transferred to grow
on 15N -derived ammonia, 15N-labeled ([m+1]) glutamate, aspartate and leucine accounted for
2
88%–90% of the corresponding amino acids in cell protein (Figure 3a and Supplementary Figure
12). This indicates that the protein component of M. sanxanigenens cell protein is synthesized from
N -derived ammonia. In the 3-L fed-batch fermentation (Supplementary Figure 13), the ammonia
2
stock was derived from the PEC synthesis process, while O was continuously supplied via
2
external aeration from an air compressor. By maintaining the ammonia concentration below 140
mg NH +-N L−1 (Figure 3b and Supplementary Table 1), pH within 7−7.3 and dissolved oxygen
4
(DO) above 10%, cell growth proceeded without limitations from oxygen transfer or by-product
accumulation. Detailed fermentation control parameters including gas flow rate (vvm), CH :O
4 2
ratio, pressure, and k a are provided in the Supplementary Information (Methods). It should be
L S
noted that no acids or growth-inhibiting by-products were dSetected in the culture broth of M.
sanxanigenens SO01, and rapid cell growth was observed after 12 h (Figure 3c), with a maximum
specific growth rate of 0.156 h−1 achieved (SupplemePntary Table 2).

The dry cell weight (DCW) and cell protein productivity reached 5.30 g L−1 and 147.2 mg L−1 h−1
(Figure 3d and Supplementary Figure 14e), representing a 5-fold and 18-fold increase compared
to the previously reported values,15 respectively. In total, 32.5 mmol of ammonia was consumed
per liter of broth, corresponding to the nitrogen demand of the culture. Additionally, cumulative
O
2
uptake was calculaAted by integrating the oxygen uptake rate (OUR) over time, reaching 520
mmol per liter of broth (Supplementary Figure 14c and Supplementary Note 9). Here, the basis
and units for normalizing nitrogen source utilization versus O uptake are defined as mg of N and
2
mmol of O consumed per gram of cell protein produced, respectively. When ammonia and O₂
2
uptake were normalized to DCW, the results showed that 85.8 mg NH -N and 98.1 mmol O were
3 2
consumed to produce 1 gram of cell protein. The cell protein yield on methane reached 0.97 g g−1
at 36 h, calculated as DCW (5.30 g L−1) divided by methane consumption (5.47 g L−1)
(Supplementary Table 3, equation 1), which aligns with previous findings.21 The cell protein yield
on ammonia-N was 11.65 g g−1 (Supplementary Table 4, equation 2).

To demonstrate the application value of our recombinant strain M. sanxanigenens SO01, a direct
comparison was performed between nitrate- and ammonia-utilizing bioprocesses under identical
conditions, with the total nitrogen supply uniformly set at 455 mg N per liter of broth for both
systems. For the ammonia-utilizing bioprocess, the maximum OD , peak methane uptake rate
600
(CUR), and highest DCW were 17.68, 33.60 mmol L−1 h−1, and 5.30 g L−1 (Figure 3c−d and
Supplementary Figure 14a), respectively. During cultivation, the total methane uptake reached
5.47 g L−1 with complete ammonia depletion (Figure 3b and Supplementary Table 3). These results
corresponded to methane- and nitrogen-based cell protein yields of 0.97 g DCW g−1 CH and 11.65
4
g DCW g−1 N, respectively (Supplementary Tables 3−4, equation 1−2). On the other hand, the
peak CUR was higher at 42.37 mmol L−1 h−1 in nitrate-utilizing bioprocess (Supplementary Figure
14b), accompanied by a total methane uptake of 7.42 g L−1 and a DCW of 6.94 g L−1
(Supplementary Figure 14f). The corresponding metPhane- and nitrogen-based yields were 0.94 g
DCW g−1 CH and 15.25 g DCW g−1 N (SuppleNmentary Table 3).
4

We further characterized the cell protLein of the engineered M. sanxanigenens strain. The protein
and polysaccharide contents reached 347 and 429 mg g−1 DCW, respectively, collectively
accounting for approximately 70%–80% of the DCW (Figure 3d). The isoleucine and leucine
contents of the methanotrophic cell protein (MCP) were 94.7% and 93.4% of those in whole egg,
while the valine content was comparable to that in whole egg and cow milk (Figure 3e and
Supplementary Figure 15).22 The branched-chain amino acid (BCAA) content of MCP was 10.9%
higher than that in fishmeal and closely matched that of whole egg (Figure 3f).23 Additionally, the
essential amino acid (EAA) content of MCP reached 107.1% of that in fishmeal. These results
indicate that the nutritional value of MCP is comparable to that of fishmeal, whole egg, and cow
milk. A minor yet significant intracellular peptide mixture was also identified (inset of Figure 3f),
which was reported to exhibit anti-inflammation effects and protect animal intestinal microbiota.24
Furthermore, the safety of this nutritional cell protein was verified, with an endotoxin level below
0.02 EU mL−1. Thus, our methanotrophic cell protein contains diverse nutrients and bioactive
peptides, highlighting its potential application as a functional feed additive.

Probiotic effects of nutritional cell protein on gut health
Recent studies have emphasized the promising benefits of MCP in promoting fish gut health. Here,
we employed a dextran sodium sulfate (DSS)-induced colitis mouse model, which adequately
simulated key pathological features of intestinal inflammation (relevant to human intestinal
disorders), including intestinal barrier disruption, inflammatory response, and gut microbiota
dysbiosis. The MCP was orally administered to evaluate its efficacy in ameliorating DSS-induced
colitis (Supplementary Figure 16 and Supplementary Figure 17a). Compared to the model group
(“+DSS” group), colitis mice treated with MCP (“+DSS,MCSP” group) exhibited significantly
reduced body weight loss (Figure 4a), lower disease activity index (DAI) score (Figure 4b and
Supplementary Table 5), and less colon shortening P(Figure 4c and Supplementary Figure 17c),
thereby contributing to a higher survival rate (SNupplementary Figure 17b).

The maintenance of the intestinal baLrrier integrity is critical for gut health.25 MCP treatment
mitigated DSS-induced structural integrity disruption of colonic tissue, as evidenced by reducing
mucosal ulceration, crypt damage, and inflammatory cell infiltration (Figure 4d, Supplementary
Table 6 and SupplemenAtary Data 2). Notably, the tight junction protein Occludin, which is essential
for maintaining the intestinal epithelial barrier, was markedly restored upon treatment with the
MCP (Figure 4e and Supplementary Figure 17d). Furthermore, the MCP demonstrated a
significant inhibitory effect on the protein expression of pro-inflammatory interleukin-6 (IL-6) and
tumor necrosis factor α (TNF-α) (Figure 4f and Supplementary Figure 18a–c), and restored the
expression of anti-inflammatory interleukin-10 (IL-10) compared to mice with colitis (Figure 4g
and Supplementary Figure 18d). These findings indicated that administration of MCP helped to
alleviate colitis by restoring intestinal barrier and attenuating inflammations in mice.

Nutritional cell protein protects intestinal health by modulating microenvironment
As MCP produced from CH and N -derived ammonia effectively ameliorated the inflammatory
4 2
bowel disease symptoms in mice, 16s rRNA gene sequencing was conducted to assess the effect
of MCP on intestinal microbiota (Supplementary Figure 19a). Administration of MCP significantly
increased the gut microbiota diversity in mice with colitis (Figure 5a–b and Supplementary Figure
19b–c). Additionally, the microbiota of mice treated with the MCP closely resembled that of the
healthy control group (Supplementary Figure 19d and Supplementary Note 10).

DSS treatment led to severe dysbiosis of the mice gut microbiota, which was effectively reversed
by MCP administration (Supplementary Figure 20 and Supplementary Note 11). MCP gavage
remarkably reduced the abundance of Campylobacterota, a phSylum associated with inflammation
(Figure 5c and Supplementary Figure 20b). Pathogenic genera, including Klebsiella, Enterocloster,
Muribaculum, and Helicobacter, proliferated in micPe with DSS-induced colitis, whereas MCP
treatment inhibited these pathogens and incrNeased the abundance of short-chain fatty acid-
producing probiotics, such as Alloprevote lla and Kineothrix (Supplementary Figure 20d and
Supplementary Figure 21a). At the sLpecies level, we observed a proportional rise in the anti-
inflammatory bacteria Faecalibaculum rodentium and Odoribacter laneu, while the disease-
related Helicobacter ganmani was suppressed following MCP intake (Supplementary Figure 21b
and Supplementary NoAte 12).

The recovery of the microbiota diversity may improve the distribution of metabolites and thus
contribute to the maintenance of the intestinal homeostasis. We conducted further investigations
on fecal metabolites in mice (Supplementary Figure 22–23 and Supplementary Note 13–14).
Treatment with the MCP resulted in a notable increase in prebiotic excretion including
taurochenodeoxycholic acid (TCDCA) and suberic acid, compared to the model group (Figure 5d).
TCDCA, previously reported to alleviate intestinal inflammation,26 exhibited the highest area
under the curve in a receiver operating characteristic analysis (Supplementary Figure 24 and
Supplementary Note 15). Additionally, suberic acid level significantly decreased in mice with
colitis, and was markedly elevated after MCP administration (Supplementary Table 7 and
Supplementary Data 3). This compound is highly correlated with enrichments of gut beneficial
bacteria.27 Therefore, these two metabolites were speculated as the biomarkers for attenuation of
mouse colitis through MCP treatment. To sum up, these results highlight that oral administration
of MCP assisted in mitigating mouse colitis progression by positively modulating intestinal
microbiota and enhancing prebiotics excretion.

Additionally, it is noteworthy that MCP treatment also benefits healthy mice by protecting the gut
microenvironment. This was evidenced by a boosted expression of Occludin with MCP
administration (Figure 4e, “−DSS,MCP” group versus “S−DSS” group). Furthermore, the
abundance of 11 bacterial taxa significantly increased among mice treated with MCP, including
the probiotic Lactobacillaceae, Lacticaseibacillus, anPd Prevotellaceae (Figure 5e). The MCP also
dramatically enhanced the excretion of beneficiaNl metabolites, including isovaleric acid, L-alanine,
lithocholic acid acetate, and myristic acid, in comparison to the normal mice (“−DSS” group)
(Figure 5f). Overall, our results suggesLt the potential use of MCP as a nutritional additive to prevent
inflammatory bowel disease in healthy animals.

Methods
Animal ethics statement
Mouse experimental protocols in the present study were approved by the Animal Ethics Committee
of Northwestern University (approval number: NWU-AWC-20240803M).

Strain and culture conditions
The bacterial strains used in this study are listed in Supplementary Table 8. M. sanxanigenens
NG09 was reserved in China General Microbiological Culture Collection Center (CGMCC No.
30951). Wild type and engineered M. sanxanigenens strains were grown in modified nitrate
mineral salt (NMS) medium, which comprised 1.000 g L−1 MgSO · 7H O, 0.013 g L−1
4 2
CaCl · 2H O, 1.000 g L−1 KNO , 0.109 g L−1 KH PO , 0.215 g L−1 Na HPO , and 2 mL L−1 trace
2 2 3 2 4 2 4
element solution.37 Frozen stocks of methanotroph strains were stored in −80 oC. For revival of
methanotroph strains, stocks were spread on the agar plates (NMS medium containing 1.5% agar
powder). The agar plates were then incubated in sealed jars filled with a gas mixture of 20% CH
S 4
and 80% air (v/v). A loop of colonies from the agar plate was Sinoculated into a 250-mL glass vial
containing 50 mL of NMS medium. The vials were sealed with rubber stoppers, followed by the
injection of 50 mL of CH into each vial. The vialsP were then incubated at 30 oC with constant
4
shaking at 200 rpm. Sampling was conducteNd at 12-h intervals, with CH freshly injected
4
immediately after each sampling event. Wh en the cell density (reflected by optical density at 600
nm, OD
600
) reached 1.5−2.0, it was inLoculated (10%) to the ammonia mineral salt (AMS) medium
to adapt to the change of nitrogen source, in which 1.00 g L−1 nitrate was replaced by 0.55 g L−1
NH Cl. After OD increased to 1.5−2.0, the seed culture was transferred (10%) to another fresh
4 600
AMS medium to evaluAate cell growth. Three biologically independent replicates were performed
for each group of methanotrophic bacterium cultivation.

To monitor the extracellular ammonia and nitrite concentrations, cultures were pelleted, and the
resulting supernatants were assayed by the Ammonia TNTplus Vial Test Kit (TNT-831, HACH)
or Nitrite TNTplus Vial Test (TNT-840, HACH), which can obtain results within 15 minutes. These
samples were measured by the Spectrophotometer DR3900 (HACH). The extracellular
hydroxylamine was detected by the colorimetric method,38 modified as follows: 1) 200 μL of
culture supernatant was mixed with 1.3 mL of 6% sodium acetate, 200 μL of 1% p-aminobenzene
sulfonic acid and 100 μL of 1.3% iodine (solubilized by glacial acetic acid); 2) 40 μL of 0.4 M
sodium thiosulfate was added to the mixture to react with the excess iodine; 3) 50 μL of 0.6% α-
naphthylamine was added to the mixture, and the optical density at the wave length of 520 nm
(OD ) was measured; 4) hydroxylamine concentration was determined using a standard curve
520
generated with hydroxylamine hydrochloride over the range of 20−200 μM). The hydroxylamine
concentration of samples was measured by Spectrophotometer TU1810 (Beijing Purkinje
GENERAL Instrument). For each group of methanotrophic bacterium cultivation, these assays
were all performed in three biologically independent replicates.

Transcriptomic analysis
M. sanxanigenens NG09 was grown in 500-mL spinner bottleSs to obtain sufficient cell mass for
RNA sequencing (RNA-seq).3 To compare the transcriptional differences of cell growth on
ammonia versus nitrate, 1.00 g L−1 nitrate in NMS mPedium was replaced by 0.55 g L−1 NH Cl.
4
Cell mass was collected at OD 2.0−2.5, and Nthe pellets were quickly frozen by liquid nitrogen
600
and stored in −80 oC before RNA-Seq.3 Ag ilent 2100/2200 Bioanalyzer (Agilent Technologies),
NanoDrop (Thermo Fisher Scientific ILnc.), Cutadapt (v 1.9.1) and Bowtie2 (v 2.2.6) were used for
quality control. NovaSeq 6000 (Illumina), HTSeq (v 0.6.1p1) and GOSeq (v 1.34.1) were used for
data analysis. Two biologically independent replicates were performed and analyzed for each
group of methanotropAhic bacterium cultivation. The whole genome sequencing data of M.
sanxanigenens NG09 have been deposited in National Center for Biotechnology Information
(NCBI) under accession code JBOBHP000000000
[https://www.ncbi.nlm.nih.gov/nuccore/JBOBHP000000000.1/]. The raw RNA-Seq data of
methanotrophic culture grown on ammonia versus on nitrate have been deposited in NCBI under
accession code PRJNA1266088 [https://www.ncbi.nlm.nih.gov/bioproject/1266088].

Genetic manipulations
Primers was designed by Snapgene (v 4.3.6.0), and was prepared by Tsingke Biotech. In order to
establish a controllable gene overexpression, the araC-P regulatory system was introduced into
BAD
the plasmid pAWP89 (constructed for methanotrophic cell factories by Puri et al., as described in
a previous study).19,39 By replacing the native tac promoter with the araBAD promoter (PCR
amplified from pBBR1-MCS5), a new plasmid pAPA24 was constructed (Supplementary Table 8).
The pAPA24 backbone was PCR amplified using the primers P89-F and bad89-R (Supplementary
Data 4). The target gene was incorporated into the pAPA24 backbone, and the resulting
recombinant plasmid was transformed into M. sanxanigenens NG09 according to a standard
procedure.40 The recombinant strain was selected on NMS plate containing 50 μg mL−1 kanamycin.
The gene expression was induced by adding 0−0.5 g L−1 of L-arabinose in the culture media.19
Gene knockout was conducted according to a standard proceduSre.40 Taking deletion of amtB as an
example. Upstream and downstream of amtB (both 1000 bp), and gentamycin-resistant gene were
PCR amplified by using the primers UpamtB-F, UpPamtB-R-t, DoamtB-F-t, DoamtB-R, GmR-F
and GmR-R. The knockout complex was PCR aNssembled and amplified, and was then transformed
into M. sanxanigenens by electroporation. The recombinant strain was selected on NMS plate
containing 10 μg mL−1 gentamicin.

LC-MS analysis of 15N labeled amino acids
Protein samples (two bAiologically independent replicates) were subjected to acid hydrolysis (6 N
HCl, 110 ℃,24 h) to liberate free amino acids. The hydrolysate supernatants (1 mL) were dried
under a gentle stream of nitrogen and reconstituted in 50 uL of acetonitrile: water (1:1, v/v).
Metabolic flux analysis was performed using a Thermo ScientificQ Exactive HF-X hybrid
quadrupole-Orbitrap mass spectrometer coupled to a UHPLC system. Chromatographic separation
was achieved on an ACQUITY UPLC BEH Amide column (2.1 mm x 100 mm, 1.7 m; Waters)
maintained at 30℃. The mobile phase consisted of ammonium acetate buffer (solvent A) and
acetonitrile (solvent B). The gradient elution was performed at a flow rate of 0.4 mL mL–1 as
follows: 0–0.5 min, 95% B; 0.5–7 min, 95% to 65% B; 7-8 min, 65% to 50% B; 8–9 min, 50% B;
9–9.1 min 50% to 95% B; and 9.1–12 min, 95% B.

Mass spectrometry data were acquired in electrospray ionization (ESI) mode with the following
source parameters: spray voltage, 2.5 kV; capillary temperature, 325℃; auxiliary gas heater
temperature, 300 ℃; sheath gas flow rate, 30 arbitrary units; and auxiliary gas flow rate, 10
arbitrary units. MS scans were acquired over a mass range of m/z 70-1050 with a resolution of
60,000, followed by data-dependent MS/MS (Top N = 8) with an isolation window of 1.5 m/z and
normalized collision energy of 30. Data acquisition and peak integration were performed using X
calibur software (v 4.0.27.19, Thermo Fisher Scientific). Correction for raw data of natural isotope
abundance was performed.41

Fed-batch cultivations
Two feeding scenarios were examined in spinner boPttle culture. In the high-dose (HD) scenario,
when the ammonia concentration decreased toN 5−10 mM (70−140 mg-N L−1), 1.50 mL NH Cl
4
stock solution was fed into the 500-mL spi nner bottles. In the middle-dose (MD) scenario, when
the ammonia concentration fell belowL 5 mM (70 mg-N L−1), 0.75 mL NH
4
Cl stock solution was
fed. Three biologically independent replicates were performed for each group of fed-batch
cultivations.

The ammonia stream produced from the photo-electrocatalytic ammonia synthesis. The NH Cl
4
used in the fermentation process was of high purity, and no additional contaminants were
introduced after preparation.5 To minimize the risk of trace metals or chloride ions, the PEC
catalysts and neutralization agents were carefully selected, and gas stripping and vacuum
distillation methods were applied to purify the ammonia solution derived from PEC synthesis,42
resulting in the production of a pure NH Cl stock for fed-batch fermentation.
4

The 3-L fermenters were loaded with 1.8 L AMS medium containing 0.55 g L−1 NH Cl. After
4
growing for 24−30 hours, 200 mL seed culture of M. sanxanigenens SO01 in the spinner bottle
was inoculated into each 3-L fermenter. The pH and temperature were controlled at 7−8 and 30±0.1
oC, respectively. The gas flow rate was set at 1 L min−1, resulting in a gas/liquid ratio of 0.5 vvm.
Air was supplied by air compressor. CH and air was pre-mixed at the volumetric ratio of 1:4 (e.g.,
4
CH :O = 1:0.84), and the CH and O content in the inlet gas were fixed at 20% and 16.8%,
4 2 4 2
respectively. CH , O and CO fractions in the off-gas of Fermenter 1 were monitored by gas
4 2 2
analyzers, and CH and O uptake rate (CUR and OUR respectively) were calculated.3 The
4 2
fermenter headspace was controlled at ambient pressure, and antifoam DF-104 was added when
the foam level exceeded 6/7 of the fermenter volume to prevent excessive foaming. Under the
given conditions (gas/liquid ratio of 0.5 vvm, maximum agitation speed of 1000 rpm), the O -
liquid mass transfer coefficient (k a ) was measured to be 109S.98 h−1 using the dynamic method.43
L O2
During the exponential growth period, the dissolved oPxygen was maintained within 5-40% to avoid
growth ceasing caused by oxygen depletion oNr excess oxygen supply, by mildly adjusting the
agitation speed (increased by 50−100 rpm w hen DO drops to 5%−10%). Additionally, the purified
NH
4
Cl stock was supplemented into feLrmenters to support growth demand (Supplementary Figure
12). When the supernatant ammonia was below 3 mM, 2.5-mL of the purified NH Cl stock was
4
added to reach a 5-mM increase on NH + concentration (Supplementary Table 1), and thereby the
4
NH
4
+-N concentrationA was controlled below 140 mg NH
4
+-N L−1 to minimize ammonia-induced
toxicity. After 24 h, dry cell weight (DCW) was measured each 6 h.

Likewise, the nitrate-fed-batch fermentation was performed in comparison to ammonia-fed-batch
fermentation. The initial NMS medium containing 1 g L−1 KNO , and additional 22.5 mM KNO
3 3
was fed into the fermenter to achieve the dose of 455 mg N per liter of broth (the same with
ammonia-fed-batch fermentation). The extracellular nitrate was monitored by the colorimetric
method.3 The supernatant of the fermentation broth was treated with 3 volumes of 95% (v/v)
ethanol, and the mixture was then centrifuged to pellet the precipitates. Subsequently, the optical
density of the resulting supernatant was measured at 220 nm and 270 nm, and the nitrate
concentration was quantified by referencing a pre-established standard curve.

We performed calculations using the equations below:
Cell protein yield on methane = DCW/(cumulative CH consumption) (1)
4
Cell protein yield on nitrogen = DCW/(cumulative NH -N or NO −-N uptake) (2)
3 3

Determination of nutritional compounds
The Kjeldahl nitrogen content of MCP was measured by the Kjeldahl method, and the crude
protein content was calculated by multiplying the Kjeldahl nitrogen content and the factor 6.25.
The carbohydrate content of MCP were detected by thSe glycogen kit (TY-2-Y, Comin
Biotechnology).3 To analyze the peptide mixture in MCP lysate, cell protein was resuspended in
deionized water (2 g L−1) and then sonicated under icPe incubation. The molecular size distribution
of peptides was analyzed by 18% SDS-PAGE gNel prepared by the ultra-low molecular weight Kit
(RTD6122, Real-Times (Beijing)), and w as imaged by an automatic in-gel analysis system
(ChampGel®6000, SinSage TechnoloLgy Co., Ltd.). To verify the existence of endotoxins, MCP
lysate above was assayed by ToxinSensor™ Chromogenic LAL Endotoxin Assay Kit (L00350,
Genescript). For each group of methanotrophic bacterium cultivation, these assays were all
performed in three bioAlogically independent replicates.

MCP obtained from 3-L fed-batch fermentation was pretreated before analyzing amino acids. 20–
50 mg of MCP was mixed with 10 mL of 6 M HCl, and the mixture was transferred into hydrolysis
tubes. The tubes were purged with nitrogen gas for 30 s, and they were then sealed and hydrolyzed
at 110 °C for 22–24 h. After cooling to room temperature, the hydrolysate was filtered through a
0.45 µm membrane and diluted to 50 mL. A 2 mL aliquot was evaporated to dryness at 85 °C twice
to remove residual acid. It was then reconstituted in 1 mL of sodium citrate buffer, and was passed
through a 0.22 µm filter prior to analysis. Calibration standards were prepared using an Amino
Acid Standard (AAS18, Sigma) diluted by 5-fold with sodium citrate buffer to a final concentration
of 0.25 µmol mL–1 for cystine and 0.5 µmol mL–1 for other amino acids.

Amino acid profiles of MCP were detected by a Biochrom 30+ Amino Acid Analyzer (Biochrom,
UK) equipped with a Na-type cation exchange column (200 mm × 4.6 mm, 8 µm). The mobile
phase consisted of a stepwise gradient of citrate buffers (pH 3.2, 4.25, and 6.45) delivered at a flow
rate of 25 mL h–1, with the column temperature programmed from 55 °C to 77 °C. Post-column
derivatization was carried out using ninhydrin (10 mL h–1) in a reaction coil at 138 °C, followed
by spectrophotometric detection at 570 nm and 440 nm. The instrument limit of detection was
determined to be approximately 2.5 pmol (S/N = 2) with a peak area reproducibility (CV) lower
than 1.0%.

Pasteurization treatment
To promote the release of nutritional compounNds, 2.5 mg of MCP was resuspended by 200 μL
sterile phosphate buffer solution, and was i ncubated under 70 °C for 30 min.35 To simulate mice
gastric digestion, 750 μL of the artifLicial gastric juice (pH 1.0) was added into the pasteurized
mixture, and then incubated under 37 °C and 120 rpm for 6 h. The mixtures were pelleted, and the
supernatants were assayed by the Total Nitrogen Kit (2714100-CN, HACH). Protein release
percentages can be obAtained by comparing with the baseline, which contained 2.5 mg MCP, 200
μL PBS and 750 μL artificial gastric juice, and was resuspended and immediately assayed by the
kit.

Animal experiment
Male 8-week-old C57BL/6J mice weighing between 20 and 25 g were purchased from Shaanxi
Pharmaceutical Medical Biotechnology Co., Ltd. Prior to the commencement of the experiments,
the mice were housed for one week in a standard setup consisting of a temperature of 25 ± 2 °C, a
humidity of 55 ± 5%, and a 12-h light/dark cycle.44

An extensive review of intervention studies involving microbial products in animal models was
conducted,45,46 and dose-ranging studies were performed. Ultimately, 100 mg kg−1 day−1 of
methanotrophic cell protein was selected as the experimental dosage. (Supplementary Figure 17a).
Following a seven-day period of domestication, the mice were randomly divided into four groups
(n=12). The groups were as follows: (1) “−DSS” group: treated with pure water and saline. (2)
“+DSS” group: treated with 2.0% DSS for 7 days and saline for 10 days. (3) “+DSS,MCP” group:
treated with 2.0% DSS for 7 days and MCP (100 mg kg−1) for 10 days concurrently. (4)
“−DSS,MCP” group: treated with MCP (100 mg kg−1) for 10 days. Disease activity index (DAI) of the mice was daily monitored, based on the criteria as mentioned before (Supplementary Table
6).6 The survival rate of four groups were given in SupplemenStary Figure 17b.

Histopathological and biochemical analysis
The colon tissues were embedded in paraffin anNd sectioned at a thickness of 5 μm for hematoxylin
and eosin staining. The expression levels o f IL-10, IL-6, and TNF-α in serum and colon tissue
were quantified using enzyme-linkLed immunosorbent assay kits (Shanghai, China). The
preparation of colonic proteins is conducted using a mixture of radioimmunoprecipitation assay
(RIPA) buffer and protease inhibitors. For western blotting, primary antibodies against Occludin
(Cat No. 27260-1-AAP, dilution 1: 35000) and glyceraldehyde-3-phosphate dehydrogenase
(GAPDH, Cat No. 10494-1-AP, dilution 1: 30000) were purchased from Proteintech Group, Inc.
The second antibody (Cat No. A21020 dilution 1: 20000) was purchased from Abbkine Scientific
Co., Ltd.47,48 The relative expression of Occludin was normalized by GAPDH. For each group of
mice, western blotting was performed in six biologically independent replicates.

16S rRNA and metabolomics analysis
Mouse fecal samples were promptly frozen in liquid nitrogen and then stored at −80 °C. These
samples were transported (protected by dry ice) to BGI Genomics Co., Ltd. for further analyses.
Total DNA was extracted from fecal samples using a commercial kit and evaluated using a
NanoDrop ND-1,000 spectrophotometer and agarose gel electrophoresis, respectively. PCR
amplification was performed using specific primers. The sequencing results are then subjected to
a verification process in accordance with the established guidelines for library construction on the
Illumina MiSeq platform. Qsep100 (Bioptic), Qubit3.0 Fluorometer (Invitrogen) and DNBSEQ-
G400 (BGI) were used for quality control and sequencing. Cutadapt (v 1.9.1), Bowtie2 (v 2.2.6),
HTSeq (v 0.6.1p1), GOSeq (v 1.34.1), iTools Fqtools fqcheck (v.0.25), cutadapt (v.2.6), readfq (v
1.0), Fast Length Adjustment of Short reads (v 1.2.11), USEARCH (v 7.0.1090), UCHIME (v
4.2.40), QIIME (v 1.80), RDP classifer (v 2.2) and mothur (v 1.31.2) were used for data analysis.41
The metagenomic sequencing data of mouse fecal samples have been deposited in NCBI under
accession code PRJNA1413482 [https://www.ncbi.nlm.nih.gov/bioproject/1413482].

Targeted metabolomics was applied to analyze mousPe fecal samples.41 Metabolite separation and
quantification were conducted using a Waters ACQUITY UPLC I-Class Plus (Waters, USA)
coupled with a QTRAP6500 Plus high-sen sitivity mass spectrometer (SCIEX, USA).49 A data
matrix containing information on Lmetabolite identification and quantification results was
generated using the quantification software Skyline (v 21.1.0.146). This matrix was then subjected
to further analysis to extract the desired information. Raw data of the targeted metabolomics
analysis of mouse fecaAl samples are provided in Supplementary Data 5. For each group of mice,
assays and analyses of mouse fecal samples were all performed in six biologically independent
replicates.

Statistics & Reproducibility
Mice were randomly assigned to four groups, with twelve mice housed per cage. Six mice were
randomly selected from each group for a full set of subsequent experimental analyses, and the
remaining mice were retained as reserve samples for contingency. All mice in each group received
the same treatment regimen during the experiment without any difference in feeding and
administration. Alternating analyses were performed on each group during the daytime to
eliminate any circadian rhythm and/or reagent-related biases. Histological analysis was conducted
using a blinded approach, while other experiments were analyzed by the same researcher
responsible for mouse treatment. The aforementioned randomization prevents any potential bias.
For the methanotrophic cultivations, monitoring mice during the oral administration experiment
and the relevant biochemical assays, statistical analyses were conducted using GraphPad Prism
10.6.0. Data were shown as the mean with SD. The one-way analysis of variance (ANOVA) with
Tukey’s test was used to evaluate the differences between variables (p>0.05 (not significant, NS),
*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). For transcriptomic analysis of methanotrophic
bacterium cultivation, differential expression analysis was perSformed using the DESeq2 package
in R (v 3.4.1). All statistical tests were two-sided (the default setting of DESeq2). The Benjamini-
Hochberg method was applied to adjust for multiplPe comparisons, and the false discovery rate
(FDR) was controlled at the threshold of adjusteNd p-value < 0.05.

The metagenomic sequencing data oLf mouse fecal samples was analyzed by the software R (v
3.4.1). For comparisons between two independent groups, the Wilcoxon rank-sum test was used
for non-normally distributed data. For comparisons involving multiple groups with non-normally
distributed data, the KAruskal-Wallis test was employed. Additionally, UPGMA clustering was
performed based on a Weighted Unifrac distance matrix. Details about the statistical tests are
provided in the figure legends and the source data file.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "General Comments:\nIn this study the authors present a sequence of approaches. They begin by introducing the use of air-derived ammonia for\nmethanotrophic growth, but this part relies on a previously published technology from another group rather than a new\nexperimental demonstration. They then perform transcriptomic analysis to show that ammonia is toxic, and attempt to\naddress this by overexpressing hao under arabinose induction. Although this led to higher biomass, toxicity still persisted\nabove 10 mM ammonia. Next, they conduct a fed-batch fermentation strategy in which ammonia was maintained below 10\nmM, allowing significant biomass production. The resulting biomass was characterized in terms of amino acid composition\nand tested in a DSS-induced colitis mouse model, where it showed improvement in gut microbiota.\nThe following comments need to be address for this manuscript.\nSpecific comments:\n1. Line 103: The stated PEC stoichiometry (2 NH3 : 1.5 O2) equals 0.75 mol O2 per mol NH3, not 0.9. Please correct this\nand use consistent units. If 0.9 derives from another PEC route, provide the balanced reaction and calculation.\n2. Line 105: 1:1 nitrogen source/O2 uptake” is ambiguous. Please specify the basis and units (e.g., mol N fed vs mol O2\nconsumed, per time or per biomass), and provide a numerical example. Also note that in the cited paper by Fei et. al\ncontinuous cultures are nitrate-fed, not ammonia-fed, please state this explicitly.\n3. Line 107–112: Author’s conclude PEC + air separation can supply O2 and NH3, but no calculation is shown. Please\ninclude a short example using your 3-L run data to estimate O2 uptake and N demand.\n4. Line 100: “1 mol N2 with 0.3 mol O2” is not fixed. ASUs deliver independent streams. Please clarify the technology and\nrecovery assumptions.\n5. Section “Assessing production of cell mass from CH4 and N2-derived ammonia” reads more like methods/assumptions\nthan results and cannot stand alone as a results subsection. Also, since actual runs used external aeration and NH4Cl feed,\nplease add a clarifying sentence to avoid reader confusion.\n6. Line 107: Replace balance with adequate.\n7. Line 153-163: The authors take upregulation of har and haoAB as proof of hydroxylamine stress. Please report the\nquantified fold changes explicitly, not only in the supplementary table, and if possible, include direct measurements of\nhydroxylamine accumulation. Likewise, for nitrite reductase overexpression, provide nitrite accumulation data. Although it\nwas mentioned that nitrite builds up due to enhanced hydroxylamine oxidation and hampers metabolism, I could not find\nsupporting quantitative values. Without metabolite data, these conclusions remain incomplete.\n8. For the amtA/amtB knockout, please provide ammonium uptake assays or intracellular NH4+ quantification to confirm that\ngrowth was not limited by transport.\n9. Arabinose concentrations vary between figures/sections (0.4 g/L in SI fig, 0.1 g/L in main text. Please resolve and state\nwhether arabinose affects growth beyond induction (co-substrate or stress).\n10. Please provide gas flow rate (vvm), CH4:O2 ratio, pressure, antifoam policy, and kLa, together with full gas mass\nbalances (CH4/O2 in, CO2 out). The reported Yx/CH4 = 1.09 g/g is unusually high, if possible please include raw\ncalculations.\n11. In fermentation, feeding was stopped at around 30h due to cell decline. Please provide ammonia and hydroxylamine\nprofiles, pH, DO, and residuals to justify the <10 mM control band, and discuss whether oxygen transfer or by-products\nceased the cell growth.\n12. Endotoxin is reported as <0.02 EU/mL, but PEC catalysts and neutralization may introduce trace metals or chloride.\nPlease provide data for (Fe, Ni, Cu, Bi, V) and Cl⁻ levels for the NH4Cl stock and final broth, as these are critical for feed\napplications.\n13. Many supplementary figures can be combined together and can be labeled as A-B etc. for instance S. figure 9-11.\n14. Line 213: The 1:1 protein:polysaccharide ratio should be supported with g/gDCW values.\n15. Line 218: In Figure 3e–f, compare amino acid profiles statistically to fishmeal, egg, and milk; currently “comparable” is\nqualitative.\n16. Figures 5c and 5e show relative abundance changes; please include % values and p-values.\n17. Supplementary Figure 25b: quantify fold changes of Klebsiella, Helicobacter, Alloprevotella.\n18. Line 296-299: Clarify whether other metabolites (isovaleric acid, alanine, lithocholic acid acetate, myristic acid) were\nsignificantly different after multiple-testing correction.\n19. The Discussion section highlights 5-fold higher DCW and 18-fold productivity, but please contextualize with earlier\nindustrial methanotroph SCP benchmarks (e.g., Methylococcus capsulatus data).\n20. The claim of “replacing antibiotics and vaccines with feed ingredients” does not seem appropriate. Please change the\nphrase.", "reviewer_4": "This study makes important contributions by developing a workable combined chemical-biological method to convert\nscattered methane into functional animal nutrients on-site: it successfully addresses the key problem of methanotrophic\nbacteria using ammonia inefficiently, and this solution directly boosts cell production. The study also verifies that the \"gas-to-\nprotein\" process works, and further shows the microbial nutrient helps improve DSS-induced colitis in mice, specifically by\nstrengthening the intestinal barrier, maintaining the balance of gut bacteria, and promoting the production of gut metabolites.\nThese findings lay a solid foundation for the application of methane-derived microbial cell proteins as value-added feed\nadditives. Given the novelty of the work and its important findings, I would recommend accepting the manuscript for\npublication after addressing the following minor revisions.\n1.Explain the reason for choosing the DSS-induced colitis mouse model to test how the cell mass from methanotrophic\nbacteria helps improve gut health. Explain why this model is suitable for checking whether the tested material can work as a\nprobiotic.\n2.The current way to analyze colon tissue (looking at its structure under a microscope) only uses visual checks to describe\nfeatures, which can lead to personal bias when judging key issues (e.g., stomach lining sores, intestinal gland damage,\ninflammatory cell buildup). Use a set standard scoring method to measure how much the colon tissue is damaged. Also, mark the damaged areas in Figure 4d so they’re easier to see.\n3.Provide more discussion about how the specific gut bacteria found relate to the gut’s metabolic products (e.g., TCDCA,\nsuberic acid). This will better support the proposed reasons behind how the material improves gut health.\n4.Show how the special traits of the cell mass production process (the early steps) connect to the observed benefits for gut\nhealth. This link is needed to make the study’s story complete and logical.\n5.To help readers fully understand and repeat the statistical checks of gut bacteria and metabolic products (e.g., data in\nSupplementary Figure 21−29), authors should include the necessary descriptive details in the Supplementary Information\nFile.\n6.In Methods section, clearly state how much of the test substance was used in the animal experiments. Also, give detailed\ninformation on how this amount was decided—this is important to make sure the study can be repeated and to help readers\nunderstand how the amount used affects the results.\n7.Minor concerns:\n1) Clarify the distinction between “ammonia” and “ammonium”, as the manuscript currently confuses these two substances.\n2) Replace the term “M. sanxanigenens cell mass” with “methanotrophic cell protein”, which is used to describe the\nmicrobial nutrient synthesized by methanotrophic bacteria. This will be more appropriate in this context and helps readers better\nunderstand the product’s characteristics and functions.\n3) Correct formatting inconsistencies in the manuscript: On page 13, line 339: Adjust “(TCDCA)” to “TCDCA”; In the legend\nof Figure 4e: Capitalize “occludin” to “Occludin”."}, "author_response": "We appreciate reviewers’ insightful feedback and have meticulously reviewed the manuscript to ensure clarity\nand accuracy. Thank you for your constructive remarks. The revision sentences were marked in blue in the\nmanuscript.\nReviewer #1: ... [Author response text for Reviewer 1 and 4 omitted for brevity in this example, but would be included here in full]"}, {"round": 2, "reviewer_comments": {"reviewer_1": "In accordance with reviewers' comments, the authors have successfully addressed the comments and revised manuscript\nwell. I'd like to recommend that the revised manuscript can be accepted as is for publication.", "reviewer_4": "I have reviewed the revised manuscript and the authors’ response. All my concerns have been adequately addressed, and I\nrecommend the acceptance of this manuscript for publication."}, "author_response": "We thank the reviewer for the positive comments on our work.\nResponse: It is our great pleasure to receive this positive comment from the Reviewer. Thank you!\nWe highly appreciate the editorial office’ thorough reading and comments/questions about our Manuscript!"}]

Decision letter