Metabolome Analysis of Constituents in Membrane Vesicles for Clostridium thermocellum Growth Stimulation.

The cultivation of the cellulolytic bacterium, Clostridium thermocellum, can have cost-effective cellulosic biomass utilizations, such as consolidated bioprocessing, simultaneous biological enzyme production and saccharification. However, these processes require a longer cultivation term of approximately 1 week. We demonstrate that constituents of the C. thermocellum membrane vesicle fraction significantly promoted the growth rate of C. thermocellum. Similarly, cell-free Bacillus subtilis broth was able to increase C. thermocellum growth rate, while several B. subtilis single-gene deletion mutants, e.g., yxeJ, yxeH, ahpC, yxdK, iolF, decreased the growth stimulation ability. Metabolome analysis revealed signal compounds for cell-cell communication in the C. thermocellum membrane vesicle fraction (ethyl 2-decenoate, ethyl 4-decenoate, and 2-dodecenoic acid) and B. subtilis broth (nicotinamide, indole-3-carboxaldehyde, urocanic acid, nopaline, and 6-paradol). These findings suggest that the constituents in membrane vesicles from C. thermocellum and B. subtilis could promote C. thermocellum growth, leading to improved efficiency of cellulosic biomass utilization.

1. Introduction

Cellulose is one of the most abundant organic materials on Earth. Bacteria that can grow on cellulose have been isolated from many environments that include soil, hot springs, cow rumen, termite gut, and the human intestinal tract [1]. Clostridium thermocellum (Acetivibrio thermocellus) [2], a Gram-positive thermophilic anaerobic soil bacterium, is a candidate for cellulosic biomass utilization. C. thermocellum completely degrades 4.4 g/L purified cellulose in one day [3]. It also degrades 65% of 5 g/L switchgrass in five days and 70% of 10 g/L corn hull in seven days [4,5].

C. thermocellum has been shown to produce 1.3% ethanol from 10% Avicel cellulose [6]. A strain of C. thermocellum multiply deleted for [FeFe] hydrogenase maturase, lactate dehydrogenase, pyruvate-formate lyase, Pfl-activating enzyme, phosphotransacetylase, and acetate kinase genes, which eliminated formate, acetate, and lactate production, and reduced H2 production, presented a titer of 2.2% ethanol from 6% Avicel cellulose [7]. The ethanol hyper-producing strain C. thermocellum I-1-B produced 2.4% ethanol from 8% cellulose [8]. A co-culture of a strain lacking the lactate dehydrogenase/phosphotransacetylase gene and Thermoanaerobacterium saccharolyticum produced 3.8% ethanol from 9.2% Avicel cellulose in 146 h [9]. These reports show that the cultivation of C. thermocellum can be simplified consolidated bioprocessing (CBP). This is a promising strategy because it eliminates the need to add lignocellulose-degrading enzymes that significantly increase the cost of biofuel production [10,11,12].

Some cellulolytic bacteria, including C. thermocellum, form carbohydrate-active enzyme (CAZyme) complexes that are termed cellulosomes [13,14,15,16]. The main product of enzymatic cellulose degradation is cellobiose, which leads to the feedback inhibition of cellulosomes. Supplementation with β-glucosidase (BGL) leads to the hydrolysis of cellobiose into form two glucose molecules, thereby resolving the feedback inhibition. C. thermocellum preferentially utilizes cellooligosaccharide, and glucose tends to accumulate in the culture broth [17]. Supplementation with purified BGL increased glucose production by C. thermocellum from 10% cellulose or 12% alkali pretreated rice straw by approximately 7.7% over 10 days [18]. This technology is referred to as biological simultaneous enzyme production and saccharification (BSES). BSES is similar to CBP, does not require the diverse CAZymes for the saccharification of cellulosic biomass.

We previously reported that C. thermocellum produces extracellular membrane vesicles (MVs) that are released into the broth [19]. MVs are produced in Gram-negative and Gram-positive bacteria. The latter possess a membrane that is overlaid by a relatively thick and resilient cell wall enriched in peptidoglycan [20,21]. MVs have been isolated from the culture supernatant of Gram-positive bacteria that include Bacillus subtilis, B. anthracis, Streptomyces coelicolor, Listeria monocytogenes, Staphylococcus aureus, Streptococcus mutans, S. pneumoniae, and Clostridium perfringens [22,23,24,25,26,27,28]. Klieve et al. reported the production of MVs by Ruminococcus spp., a cellulolytic bacterium that resides in the ovine rumen. DNA molecules ranging in size from <20 to 49 kb, and from 23 to 90 kb are attached to MVs from Ruminococcus sp. YE73 and Ruminococcus albus AR67, respectively. Thus, MVs can function as vectors for horizontal gene transfer to confer cellulolytic activity, as documented in the mutant strain Ruminococcus sp. YE71 [29]. MVs from cellulolytic Bacteroides fragilis and B. thetaiotaomicron are equipped with hydrolytic enzymes and are important in polysaccharide degradation [30,31]. MVs from Fibrobacter succinogenes are enriched with CAZymes, and intact MVs are able to degrade a broad range of hemicelluloses and pectin [32]. We have previously proposed that C. thermocellum may utilize MVs to deliver cellulosomes, which enhance the cellulolytic activity of C. thermocellum [19].

MVs contain various compounds that include DNA and RNA. These cargos are delivered to neighboring cells. MVs have several important functions related to cell–cell interactions. In Pseudomonas aeruginosa, a hydrophobic cell–cell communication signal termed Pseudomonas quinolone signal is released from the bacteria via MVs [33,34]. MVs can also serve as organic carbon sources for heterotrophs. For example, MVs derived from cyanobacteria support the growth of Alteromonas and Halomonas as the sole carbon source, indicating that MVs should be considered in the marine food web and may have important roles in the carbon flux of the ocean [35]. In Mycobacterium tuberculosis, the causative agent of tuberculosis, increased MV production in response to iron restriction has been observed [36]. These MVs contain a siderophore called mycobactin. Mycobactin can serve as an iron donor to support the growth of iron-starved M. tuberculosis.

In this study, we demonstrated that the MV fractions collected from C. thermocellum and B. subtilis can promote C. thermocellum growth. Metabolome analysis was also performed to identify the candidate compounds with the growth stimulation.

2. Materials and Methods

2.1. Strains and Culture Conditions of C. thermocellum and B. subtilis

One hundred microliters of C. thermocellum DSM 1313 (DSMZ, Braunschweig, Germany) culture was inoculated in 5 mL of CTFUD medium (3 g/L sodium citrate tribasic dehydrate, 1.3 g/L (NH4)2SO4, 1.5 g/L KH2PO4, 130 mg/L CaCl2 2H2O, 500 mg/L L-cysteine-HCl, 11.56 g/L 3-morpholinopropanesulfonic acid, 2.6 g/L MgCl2 6H2O, 1 mg/L FeSO4 7H2O, 4.5 g/L Bacto yeast extract, 1 mg/L resazurin, pH 7.0) containing 0.5% cellobiose (Tokyo Chemical Industry, Tokyo, Japan) with 16 × 125 mm Hungate tubes (Chemiglass Life Sciences, Vineland, NJ, USA), and cultured at 60 °C under anaerobic conditions with nitrogen gas [37].

B. subtilis KAO/NAIST chromosomal deletion mutants [38] and BKE genome-scale deletion mutants [39] were obtained from the National BioResource Project B. subtilis (National Institute of Genetics, Shizuoka, Japan). B. subtilis strains were aerobically cultured in Luria Bertani broth at 37 °C.

2.2. Preparation of MV Fraction of C. thermocellum

Five milliliters of C. thermocellum and B. subtilis culture was centrifuged at 10,000× g for 2 min at 4 °C, and the supernatant was filtered through a 0.22-μm syringe filter to remove cells. The filtrate was centrifuged at 179,000× g for 1 h at 4 °C and the pellet was washed twice with 2 mL of sterile phosphate-buffered saline (PBS). The pellet was resuspended in PBS and used as the MV fraction. The MV fraction was kept on ice before use.

MVs were visualized using transmission electron microscopy. Six microliter aliquots of the MV fraction was added to 300-mesh carbon and formvar-coated copper grids and incubated for 1 min. After removing the extra solution with filter paper, each specimen was stained with 2% phosphotungstic acid. The sample was observed with a JEM-1011 microscope (JEOL, Tokyo, Japan) at an accelerating voltage of 80 kV.

2.3. Growth Evaluation of C. thermocellum with MV Supplementation

One hundred microliters of C. thermocellum DSM 1313 culture was inoculated in 5 mL of CTFUD medium containing 0.5% cellobiose with the supplementation of the collected MV fraction. C. thermocellum was cultured at 60 °C under anaerobic conditions with nitrogen gas. The C. thermocellum growth was evaluated with optical density of the broth at 600 nm.

2.4. Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) Analysis of C. thermocellum MV and B. subtilis Broth

The C. thermocellum MV fraction was treated with 10 mg/L surfactin, and the filtrate obtained after ultrafiltration with Vivaspin 2-100 K (Cytiva, Marlborough, MA, USA) was used to obtain the constituents in MVs. Cell-free supernatants of B. subtilis trpC2 and trpC2 yxeJ broth were prepared by centrifugation and filtration with a 0.22-μm syringe filter. These specimens were homogenized with zirconia beads in 75% methanol, and the supernatants were collected after centrifugation at 15,000× g rpm for 10 min. The supernatants were applied to a MonoSpin C18 column (GL Science, Tokyo, Japan) and were filtered through a 0.22-μm syringe filter.

LC-MS analysis was performed on an Ultimate 3000 rapid separation LC (RSLC) and the Q Exactive system (Thermo Fisher Scientific, Waltham, MA, USA). Ultimate 3000 RSLC analysis was performed with the following parameters: column, InertSustain AQ-C18 (GL Science); column temperature, 40 °C; injection volume, 2 µL; solvent flow rate, 200 µL/min. The eluting solution was 0.1% formic acid containing 2% acetonitrile. The Q Exactive system had the following parameters: measurement time, 3–30 min; ionization method, electrospray ionization; measurement mass range, m/z: 80–1200; full scan resolution, 70,000; and MS/MS scan resolution, 17,500. The obtained data were analyzed with PowerGetBatch and MFSearcher [40]. The LC-MS analysis was performed in triplicate.

3. Results and Discussion

3.1. MV Constituents Promote C. thermocellum Growth

A previous study reported that the co-culture of the engineered C. thermocellum and T. saccharolyticum strains produced 3.8% ethanol from cellulose for 6 days [9]. C. thermocellum cultivation with BGL supplementation for 10 days reportedly produced 76.7 g/L glucose from alkali pretreated rice straw [18]. It seems that the growth rate of C. thermocellum is an important factor in improving the efficiency of CBP and BSES. In this study, we collected MVs from C. thermocellum broth (Figure S1). MVs contain various compounds, such as DNA and RNA, which function in cell–cell communication. When C. thermocellum was grown in the presence of the MV fraction, the growth rate did not change. However, when the MVs were lysed using the lipopeptide surfactin [41] the cell density of C. thermocellum had significantly increased at 24 h after the inoculation (Figure 1). The surfaction supplementation alone did not affect the C. thermocellum growth rate. The final growth yield in each sample had not changed significantly. These results suggest that the constituents in the MV fraction could promote the growth rate of C. thermocellum.

Figure 1

C. thermocellum growth stimulation by the MV constituents. C. thermcellum was cultured in CTFUD medium for 24 h with the supplementation of water, the MV fraction, or the surfactin-treated MV fraction. The cultures (a) and their optical densities (b) are shown. The experiment was duplicated. Error bars show standard error. * Student’s t-test p < 0.01.

3.2. B. subtilis Broth Promotes C. thermocellum Growth Rate

Cell-free B. subtilis broth containing MVs also promoted the C. thermocellum growth rate, similar to the C. thermocellum MV fraction (Figure S1 and Figure 2a). Again, the surfaction supplementation alone did not affect the C. thermocellum growth rate (Figure 2a). Mukamolova et al. purified the resuscitation promoting factor (Rpf) from the broth of the Gram-positive bacterium, Micrococcus luteus. The purified Rpf promoted the growth of this bacterium as well as Mycobacterium avium, M. bovis, M. kansasii, M. smegmatis, and M. tuberculosis [42]. Genes homologous to the rpf gene were found to be widespread in a number of Mycobacterium species, as well as in Gram-positive bacteria with a high GC content, such as Corynebacterium gultamicum and Streptomyces rimosus. The Rpf protein shows peptidoglycan degradation activity [43]. Shah et al. reported that muropeptide fragments released from the peptidoglycan of the Gram-positive bacterium, B. subtilis, stimulate the germination of bacterial spores. Staurosporine, which inhibits related eukaryotic kinases in bacteria, blocks muropeptide-dependent bacterial spore germination [44]. We evaluated the effect of staurosporine on C. thermocellum growth with cell-free B. subtilis broth, however no significant inhibition was observed.

Figure 2

(a) C. thermocellum growth stimulation using cell-free B. subtilis broth. C. thermcellum was cultured in CTFUD medium with surfactin-treated cell-free B. subtilis broth for 24 h. The experiment was performed in triplicate. * Student’s t-test p < 0.01. (b) C. thermocellum growth promotion effect of the broth of B. subtilis genome deletion mutants evaluated. The genotypes of the genome deletion mutants are listed in Table S1. The experiment was duplicated. (c) Evaluation of the C. thermocellum growth promotion effect of the broth of B. subtilis single-gene deletion mutants. Dark and light blue indicate significant differences compared with the effect of the parent strain (trpC2) with Student’s t-test at p < 0.01 and < 0.05, respectively. The experiment was duplicated. Error bars indicate the standard error.

We further evaluated the C. thermocellum growth promotion effect of the broth of B. subtilis genome deletion mutants [38]. All the mutants, especially six mutants in which the pdp-rocR genomic region, were deleted (MGB723, MGB773, MGB822, MGB834, MGB860, MGB874) promoted C. thermocellum growth by accelerating the growth rate (Figure 2b, Table S1). Subsequently, we evaluated the C. thermocellum growth promotion effect of 100 B. subtilis mutants in which single genes within the pdp-rocR genomic region were deleted under a trpC2 gene deletion background (Table S2) [39]. We did not find B. subtilis mutants that promoted C. thermocellum growth more than trpC2 strain as the parent strain. Contrary to our expectation, the effect of 23 B. subtilis mutants was significantly lower than that of the parent strain (Figure 2c).

Among these 23 genes, the functions of several genes have been experimentally evaluated. The asnH operon, which comprises yxbB, yxbA, yxnB, asnH, and yxaM, might be involved in the biosynthesis of asparagine [45]. The iolJ, iolG, iolF, iolE, iolC, iolB, and iolR genes in the iolABCDEFGHIJ and iolRS operon are responsible for myo-inositol catabolism involving multiple and stepwise reactions [46,47,48]. We observed a slight growth inhibition of C. thermocellum in the presence of myo-inositol, however this required a high concentration (1 mg/mL) of myo-inositol (Figure S2). YydF is predicted to be an exported and modified peptide that has antimicrobial and/or signaling properties [49,50]. YxaL, which contains a repeated pyrrolo-quinoline quinone (PQQ) domain that forms a beta-propeller structure, interacts with the DNA helicase PcrA in B. subtilis [51]. Kim et al. reported that treatment of Arabidopsis thaliana and Oryza sativa L. seeds with 1 mg/L purified YxaL was effective in improving root growth [52]. PQQ, which was first recognized as an enzyme cofactor in bacteria, displays bioactivities for various eukaryotes and prokaryotes. For many bacterial species, PQQ has growth stimulation effect and serves as a cofactor for a special class of dehydrogenases/oxidoreductases [53]. PQQ has been described as an essential growth factor for various microbes [54,55,56]. We observed a slight C. thermocellum growth promotion effect by PQQ. This effect was not enough to explain the effect of B. subtilis broth (Figure S3). More than 50 proteins are involved in B. subtilis spore coat assembly. Of these, YxeE is an inner spore coat protein [57,58]. ahpC encodes thiol-specific peroxidase that plays a role in protecting cells against oxidative stress by detoxifying peroxides [59]. Utilization of a hydroxamate siderophore, ferrioxamine, requires the FhuBGC ABC transporter together with a ferrioxamine-binding protein, YxeB [60]. A range of siderophores can act as growth factors for various previously uncultured bacteria [61]. YxdK is assumed to be a subunit of the two-component sensor histidine kinase, with its potential cognate response regulator, YxdJ [62]. Co-cultivation with B. subtilis allows the growth of Synechococcus leopoliensis CCAP1405/1 on solid media. However, the yxdK deletion mutant reportedly loses this ability [63]. The yxeK gene, which encodes FAD-dependent monooxygenase, contributes to the metabolism of S-(2-succino)cysteine to cysteine [64].

3.3. Metabolome Analysis of the Constituents in C. thermocellum MV and B. subtilis Broth

We collected the constituents in C. thermocellum MVs and analyzed them using LC-MS/MS. Among the 534 detected peaks, the intensities of seven peaks were significantly higher in the fraction where MVs had been disrupted by surfactin compared to MVs not disrupted using surfactin (Table S3). The structure of five significantly detected compounds in surfactin-treated C. thermocellum MVs specimen can be estimated by MS/MS analysis (Table 1 and Table S5).

Table 1

The constituents in C. thermocellum MVs detected by LC-MS/MS analysis.

No.	Formula	Exact Mass	Name	Database	Database ID
3203	C16H31O1N1	253.241		EX-HR2
3013	C12H22O2	198.162	Ethyl 2-decenoate	UC2	HMDB0037329
	C12H22O2	198.162	Ethyl 4-decenoate	UC2	HMDB0039220
	C12H22O2	198.162	Methyl 9-undecenoate	UC2	HMDB0037305
	C12H22O2	198.162	Methyl 10-undecenoate	UC2	HMDB0029585
	C12H22O2	198.162	Allyl nonanoate	UC2	HMDB0029763
	C12H22O2	198.162	cis-3-Hexenyl hexanoate	UC2	HMDB0033378
	C12H22O2	198.162	2-Hexenyl hexanoate	UC2	HMDB0038924
	C12H22O2	198.162	Hexyl 2E-hexenoate	UC2	HMDB0038269
	C12H22O2	198.162	Hexyl 2-methyl-3-pentenoate	UC2	HMDB0040158
	C12H22O2	198.162	Hexyl 2-methyl-4-pentenoate	UC2	HMDB0040163
	C12H22O2	198.162	1-Ethenylhexyl butanoate	UC2	HMDB0037498
	C12H22O2	198.162	2-Octenyl butyrate	UC2	HMDB0038081
	C12H22O2	198.162	cis-4-Decenyl acetate	UC2	HMDB0032214
	C12H22O2	198.162	Menthyl acetate	UC2	C00036314
	C12H22O2	198.162	Rhodinyl acetate	UC2	HMDB0037186
	C12H22O2	198.162	Citronellyl acetate	UC2	C00035564
	C12H22O2	198.162	2-Dodecenoic acid	UC2	HMDB0010729
	C12H22O2	198.162	4-dodecenoic acid	UC2	C00051284
	C12H22O2	198.162	5-dodecenoic acid	UC2	HMDB0000529
	C12H22O2	198.162	11-Dodecenoic acid	UC2	HMDB0032248
	C12H22O2	198.162	5-dodecalactone	UC2	HMDB0037742
	C12H22O2	198.162	gamma-Dodecalactone	UC2	C00030347
	C12H22O2	198.162	epsilon-Dodecalactone	UC2	HMDB0038895
	C12H22O2	198.162	alpha-Heptyl-gamma-valerolactone	UC2	HMDB0037813
	C12H22O2	198.162	4-butyl-4-hydroxyoctanoic acid lactone	UC2	HMDB0036182
	C12H22O2	198.162	2,6-Dimethyl-5-heptenal propyleneglycol acetal	UC2	HMDB0032235
	C12H22O2	198.162	citral dimethyl acetal	UC2	HMDB0040361
	C12H22O2	198.162	citronelloxyacetaldehyde	UC2	HMDB0041449
	C12H22O2	198.162	Chokol A	UC2	C00011518
	C12H22O2	198.162	Cybullol	UC2	C00013221
42	C12H4O3S1	227.988		EX-HR2
3000	C18H35O2N	297.267	Lepadin D	UC2	C00026353
	C18H35O2N	297.267	Cassine	UC2	C00002027
2834	C8H13ON	139.100	5-Pentyloxazole	UC2	HMDB0038792
	C8H13ON	139.100	4,5-Dimethyl-2-propyloxazole	UC2	HMDB0037869
	C8H13ON	139.100	4,5-Dimethyl-2-isopropyloxazole	UC2	HMDB0037871
	C8H13ON	139.100	4-Butyl-2-methyloxazole	UC2	HMDB0037855
	C8H13ON	139.100	2,4-Dimethyl-5-propyloxazole	UC2	HMDB0037868
	C8H13ON	139.100	4,5-Diethyl-2-methyloxazole	UC2	HMDB0037870
	C8H13ON	139.100	2-Pentyloxazole	UC2	HMDB0037818
	C8H13ON	139.100	7beta-Hydroxy-1-methylene-8alpha-pyrrolizidine	UC2	C00026172
	C8H13ON	139.100	2-propionyltetrahydropyridine	UC2	HMDB0034884
	C8H13ON	139.100	alpha-Phosphinylbenzyl alcohol	UC2	HMDB0029613
	C8H13ON	139.100	Supinidine	UC2	C00002120
	C8H13ON	139.100	Tropinone	UC2	C00037960

An aliphatic compound with the chemical formula C12H22O2 was specifically detected in surfactin-treated C. thermocellum MVs (Table 1). Cis-2-decenoic acid was reported to decrease persister formation and revert dormant cells to a metabolically active state. Wang et al. demonstrated that three medium-chain unsaturated fatty acid ethyl esters (ethyl trans-2-decenoate, ethyl trans-2-octenoate, and ethyl cis-4-decenoate) decreased persister formation in Escherichia coli, P. aeruginosa, and Serratia marcescens, suggesting that fatty acid ethyl esters disrupt bacterial dormancy [65].

Some aliphatic acids function as diffusible signal factors (DSFs). These include cis-11-methyl-2-dodecenoic acid from Xanthomonas campestris and cis-2-dodecenoic acid from Burkholderia cenocepacia, among others [66]. DSFs are synthesized by and interact with a diverse group of microbes, including fungi, suggesting a broad conservation of cell-cell communication among these organisms [67,68,69,70]. Mutation of the DSF biosynthesis gene in B. cenocepacia results in substantially impaired growth in minimal medium [71]. Dean et al. demonstrated that Burkholderia DSF inhibits the formation and disperses Francisella biofilms. Furthermore, Burkholderia DSF was reported to upregulate the genes involved in iron acquisition in F. novicida, which increased siderophore production [72].

Subsequently, we compared the metabolites in the broth of B. subtilis trpC2 and trpC2 yxeJ (Figure 2). Among the 3150 detected peaks, the intensities of 40 peaks were significantly higher in the broth of B. subtilis trpC2 compared to that of trpC2 yxeJ (Table S4). The structures of 32 significantly detected compounds in B. subtilis trpC2 broth were estimated by MS/MS analysis (Table 2 and Table S5). Diverse peptides were detected in B. subtilis trpC2 broth. Nicotinamide reportedly enhances growth of both Gram-negative and Gram-positive bacteria, such as M. avium, Propionibacterium acnes, S. aureus, and B. macerans [73,74,75,76]. Indole-3-carboxaldehyde was shown to efficiently inhibit biofilm formation by Vibrio cholerae O1 [77]. The utilization of urocanic acid by Pseudomonas and Aeromonas strains has been reported [78,79]. Nopaline is a carbon and nitrogen source metabolized by Agrobacterium. 6-Paradol was reported to have significant anti-adhesive activity against S. aureus [80].

Table 2

The constituents in B. subtilis trpC2 broth detected by LC-MS/MS analysis.

No.	Formula	Exact Mass	Name	Database	Database ID
1938	C12H23O8N	309.142	4-O-beta-D-Glucopyranosylfagomine	UC2	C00049954
1980	C15H39O2N7S3	445.233		EX-HR2
453	C16H30O6N6	402.223		EX-HR2
2242	C17H31O4N3	341.231	Diprotin A	UC2	C00018579
1607	C25H40O7	452.277	Briarellin P	UC2	C00044586
799	C10H20O3N2S	248.119	Valyl-Methionine	UC2	HMDB0029133
	C10H20O3N2S	248.119	Methionyl-Valine	UC2	HMDB0028986
510	C11H22O4N4	274.164	Glutaminyllysine	UC2	HMDB0028802
	C11H22O4N4	274.164	Lysyl-Gamma-glutamate	UC2	HMDB0028965
	C11H22O4N4	274.164	Lysyl-Glutamine	UC2	HMDB0028949
960	C21H40O1N3P3	443.238		EX-HR2
2575	C8H13N3P2	213.058		EX-HR2
2345	C19H29N3O4S1	395.188	V1M1F1	Pep1000
2536	C29H36O5N4	520.269	Lotusine F	UC2	C00027221
	C29H36O5N4	520.269	Nummularine S	UC2	C00029150
2237	C33H44O11	616.288	Neoazedarachin A	UC2	C00039833
	C33H44O11	616.288	YM 47524	UC2	C00016365
2633	C23H55O12N1P2	599.320		EX-HR2
2673	C46H67O2N10P1S1	854.491		EX-HR2
1271	C27H44O9	512.299	Butyrolactol B	UC2	C00016754
	C27H44O9	512.299	Integristerone B	UC2	C00048431
	C27H44O9	512.299	Platenolide B mycarose	UC2	C00018288
162	C6H6ON2	122.048	Nicotinamide	UC2	C00000209
	C6H6ON2	122.048	2-Acetylpyrazine	UC2	HMDB0031861
1710	C15H24O4N4	324.180		EX-HR2
211	C6H9O3N	143.058	SQ 26517	UC2	C00018434
	C6H9O3N	143.058	Trimethadione	UC2	HMDB0014491
	C6H9O3N	143.058	6-Oxopiperidine-2-carboxylic acid	UC2	HMDB0061705
	C6H9O3N	143.058	5-ethyl-5-methyl-2,4-oxazolidinedione	UC2	HMDB0061082
	C6H9O3N	143.058	Vinylacetylglycine	UC2	HMDB0000894
	C6H9O3N	143.058	Methyl pyroglutamate	UC2	C00051578
1258	C22H66N2P2S6	612.303		EX-HR2
994	C20H33N5O8	471.233	G2[L|I]1E1P1, G1A1V1E1P1, G1A1[L|I]1D1P1, G1T2P2, A2V1D1P1, A1S1T1P2, V1E1Q1P1, [L|I]1D1Q1P1, [L|I]1E1N1P1	Pep1000
655	C16H27N5O6	385.196	G3V1P1, G1A3P1, G1V1N1P1, A2Q1P1	Pep1000
1034	C10H16O3N2	212.116	Butabarbital	UC2	HMDB0014382
	C10H16O3N2	212.116	L-prolyl-L-proline	UC2	HMDB0011180
	C10H16O3N2	212.116	Butethal	UC2	HMDB0015442
457	C32H48O5N2S1	572.328		EX-HR2
2755	C9H7ON	145.053	Indole-3-carboxaldehyde	UC2	C00000112
	C9H7ON	145.053	2-Quinolone	UC2	C00044432
2680	C67H108O6N2S5	1196.681		EX-HR2
115	C6H6O2N2	138.043	4-Methoxylonchocarpin	UC2	HMDB0031338
	C6H6O2N2	138.043	2-Aminonicotinic acid	UC2	HMDB0061680
	C6H6O2N2	138.043	Urocanic acid	UC2	HMDB0062562
	C6H6O2N2	138.043	Nicotinamide N-oxide	UC2	HMDB0002730
2949	C11H21ON	183.162	Tecostanin	UC2	C00001984
	C11H21ON	183.162	Incarvilline	UC2	C00050294
1600	C20H57O4N9S3	583.370		EX-HR2
1727	C11H20O6N4	304.138	Nopaline	UC2	C00001548
526	C21H56O14N10P2	734.345		EX-HR2
3061	C17H26O3	278.188	1-Acetoxy-3,15-epoxygymnomitrane	UC2	C00021889
	C17H26O3	278.188	Litsealactone B	UC2	C00044889
	C17H26O3	278.188	9beta-Acetoxy-10(14)-aromadendren-4beta-ol	UC2	C00021235
	C17H26O3	278.188	Furoscrobiculin C	UC2	C00021531
	C17H26O3	278.188	[S-[R *,S *-(E)]]-6-[6-(Acetyloxy)-1,5-dimethyl-4-hexenyl]-3-methyl-2-cyclohexen-1-one	UC2	C00011679
	C17H26O3	278.188	Panaxytriol	UC2	C00030923
	C17H26O3	278.188	Panaxacol	UC2	HMDB0039251
	C17H26O3	278.188	Parahigginol C	UC2	C00049252
	C17H26O3	278.188	[1S-(1R *,2E,4R *,5R *,6E,10R *)]-3, 7, 11, 11-Tetramethylbicyclo [8.1.0]undeca-2,6-diene-4,5-diol 5-acetate	UC2	C00012427
	C17H26O3	278.188	Isoobtusilactone	UC2	C00050966
	C17H26O3	278.188	8beta-Acetoxy-9beta-hydroxyverboccidenten	UC2	C00020229
	C17H26O3	278.188	Lincomolide B	UC2	C00047968
	C17H26O3	278.188	[1S-(1R *,2E,4R *,5R *,6E,10R *)]-3, 7, 11, 11-Tetramethylbicyclo[8.1.0]undeca-2,6-diene-4,5-diol 4-acetate	UC2	C00012428
	C17H26O3	278.188	4alpha-Hydroxygymnomitryl acetate	UC2	C00021894
	C17H26O3	278.188	4-[(4E)-3-hydroxydec-4-en-1-yl]-2-methoxyphenol	UC2	HMDB0137260
	C17H26O3	278.188	Ro 09-1544	UC2	C00017230
	C17H26O3	278.188	6-Paradol	UC2	C00002764
	C17H26O3	278.188	Paralemnolin D	UC2	C00030924
	C17H26O3	278.188	Fenoksan; Fenoxan; Fenozan; Fenozan acid; Irganox 1310; Phenosan; Phenoxan; Phenozan	UC2	C00016759
	C17H26O3	278.188	[4aR-(4aalpha,5alpha,8abeta,9abeta)]-9a-Ethoxy-4a, 5, 6, 7, 8, 8a, 9, 9a-octahydro-3,4a,5-trimethyl-naphtho[2,3-b]furan-2(4H)-one	UC2	C00017405
	C17H26O3	278.188	Petasipalin B	UC2	C00020246
	C17H26O3	278.188	4-epi-7alpha,15-dihydroxypodocarp-8(14)-en-13-one;(-)-4-epi-7alpha,15-dihydroxypodocarp-8(14)-en-13-one	UC2	C00035020
	C17H26O3	278.188	3-[(Acetyloxy)methyl]-6-(1,5-dimethyl-4-hexenyl)-2-cyclohexen-1-one	UC2	C00011682
	C17H26O3	278.188	Cyclokessyl acetate	UC2	C00020354
	C17H26O3	278.188	8-Acetoxy-4-acoren-3-one	UC2	HMDB0030974
832	C12H34O3N6S3	406.185		EX-HR2

In this study, we demonstrated that constituents in membrane vesicles significantly promoted the growth rate of C. thermocellum. Additionally, the MV constituents with growth stimulation were described by LC-MS/MS analysis. These findings suggest that the constituents in membrane vesicles could promote C. thermocellum growth, leading to improved efficiency of cellulosic biomass utilization.